Mapping the Genes for Susceptibility and Response to Leishmania tropica in Mouse

Background

L. tropica can cause both cutaneous and visceral leishmaniasis in humans. Although the L. tropica-induced cutaneous disease has been long known, its potential to visceralize in humans was recognized only recently. As nothing is known about the genetics of host responses to this infection and their clinical impact, we developed an informative animal model. We described previously that the recombinant congenic strain CcS-16 carrying 12.5% genes from the resistant parental strain STS/A and 87.5% genes from the susceptible strain BALB/c is more susceptible to L. tropica than BALB/c. We used these strains to map and functionally characterize the gene-loci regulating the immune responses and pathology.

Methods

We analyzed genetics of response to L. tropica in infected F₂ hybrids between BALB/c×CcS-16. CcS-16 strain carries STS-derived segments on nine chromosomes. We genotyped these segments in the F₂ hybrid mice and tested their linkage with pathological changes and systemic immune responses.

Principal Findings

We mapped 8 Ltr (Leishmania tropica response) loci. Four loci (Ltr2, Ltr3, Ltr6 and Ltr8) exhibit independent responses to L. tropica, while Ltr1, Ltr4, Ltr5 and Ltr7 were detected only in gene-gene interactions with other Ltr loci. Ltr3 exhibits the recently discovered phenomenon of transgenerational parental effect on parasite numbers in spleen. The most precise mapping (4.07 Mb) was achieved for Ltr1 (chr.2), which controls parasite numbers in lymph nodes. Five Ltr loci co-localize with loci controlling susceptibility to L. major, three are likely L. tropica specific. Individual Ltr loci affect different subsets of responses, exhibit organ specific effects and a separate control of parasite load and organ pathology.

Conclusion

We present the first identification of genetic loci controlling susceptibility to L. tropica. The different combinations of alleles controlling various symptoms of the disease likely co-determine different manifestations of disease induced by the same pathogen in individual mice.     

Potential Reduction of Contralateral Second Breast-Cancer Risks by Prophylactic Mammary Irradiation: Validation in a Breast-Cancer-Prone Mouse Model

Background

Long-term breast-cancer survivors have a highly elevated risk (1 in 6 at 20 years) of contralateral second breast cancer. This high risk is associated with the presence of multiple pre-malignant cell clones in the contralateral breast at the time of primary breast cancer diagnosis. Mechanistic analyses suggest that a moderate dose of X-rays to the contralateral breast can kill these pre-malignant clones such that, at an appropriate Prophylactic Mammary Irradiation (PMI) dose, the long-term contralateral breast cancer risk in breast cancer survivors would be considerably decreased.

Aims

To test the predicted relationship between PMI dose and cancer risk in mammary glands that have a high risk of developing malignancies.

Methods

We tested the PMI concept using MMTV-PyVT mammary-tumor-prone mice. Mammary glands on one side of each mouse were irradiated with X-rays, while those on the other side were shielded from radiation. The unshielded mammary glands received doses of 0, 4, 8, 12 and 16Gy in 4-Gy fractions.

Results

In high-risk mammary glands exposed to radiation doses designed for PMI (12 and 16 Gy), tumor incidence rates were respectively decreased by a factor of 2.2 (95% CI, 1.1-5.0) at 12 Gy, and a factor of 3.1 (95% CI, 1.3-8.3) at 16 Gy, compared to those in the shielded glands that were exposed to very low radiation doses. The same pattern was seen for PMI-exposed mammary glands relative to zero-dose controls.

Conclusions

The pattern of cancer risk reduction by PMI was consistent with mechanistic predictions. Contralateral breast PMI may thus have promise as a spatially targeted breast-conserving option for reducing the current high risk of contralateral second breast cancers. For estrogen-receptor positive primary tumors, PMI might optimally be used concomitantly with systemically delivered chemopreventive drugs such as tamoxifen or aromatase inhibitors, while for estrogen-receptor negative tumors, PMI might be used alone.     

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan δ solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials'' morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by Stöber''s methods, which have smooth surfaces.     

An assessment of DFT methods for predicting the thermochemistry of ion-molecule reactions of group 14 elements (Si, Ge, Sn)

Experimental mass-spectrometry data on thermochemistry of methide transfer reactions (CH₃)₃M⁺ + M'(CH₃)₄ ? M(CH₃)₄?+?(CH₃)₃M'⁺ (M, M'?=?Si, Ge or Sn) and the formation energy of the [(CH₃)₃Si-CH₃-Si(CH₃)₃]⁺ complex are used as benchmarks for DFT methods (B3LYP, BMK, M06L, and ωB97XD). G2 and G3 theory methods are also used for the prediction of thermochemical data. BMK, M06L, and ωB97XD methods give the best fit to experimental data (close to chemical accuracy) as well as to G2 and G3 results, while B3LYP demonstrates poor performance. From the first three methods M06L gives the best overall result. Structures and formation energies of intermediate “mixed” [(CH₃)₃M-CH₃- M′(CH₃)₃] complexes not observed in experiment are predicted. Their structures, better described as M(CH₃)₄?[M′(CH₃)₃]⁺ complexes, explain their fast decompositions.

Splitting of Plasmon Frequency in Spherical Metal Nanoparticles in Anisotropic Medium

The problem of plasmon resonance in spherical metal nanoparticles incorporated into an anisotropic dielectric medium is studied theoretically. Analytic solution is obtained in long-wavelength approximation for the case of weak uniaxial anisotropy. It is shown that medium anisotropy causes shifts of plasmon frequency from its position in an isotropic medium, which are different for plasmons with dipole momenta parallel and perpendicular to the axis of the medium. For the parallel and perpendicular orientations, which are determined by polarization of incident light, plasmon shifts differ by a factor of 4/3. Analytic expressions for field enhancement in the vicinity of a metal nanoparticle is obtained and analyzed for the cases of noble metals. The perspectives of experimental check of the obtained results and possible applications of plasmon anisotropy in plasmonics and sensorics are discussed.     

1H, 13C, and 15N backbone resonance assignments of the L124D mutant of StAR-related lipid transfer domain protein 4 (StARD4)

Protein-mediated cholesterol trafficking is central to maintaining cholesterol homeostasis in cells. START (Steroidogenic acute regulatory protein-related lipid transfer) domains constitute a sterol and lipid binding motif and the START domain protein StARD4 typifies a small family of mammalian sterol transport proteins. StARD4 consists of a single START domain and has been reported to act as a general cholesterol transporter in cells. However, the structural basis of cholesterol uptake and transport is not well understood and no cholesterol-bound START domain structures have been reported. We have undertaken the study of cholesterol binding and transport by StARD4 using solution state NMR spectroscopy. To this end, we report nearly complete ¹H, ¹⁵N, and ¹³C backbone resonance assignments of an inactive but well behaved mutant (L124D) of StARD4.     

Leucine and HMB Differentially Modulate Proteasome System in Skeletal Muscle under Different Sarcopenic Conditions

In the present study we have compared the effects of leucine supplementation and its metabolite β-hydroxy-β-methyl butyrate (HMB) on the ubiquitin-proteasome system and the PI3K/Akt pathway during two distinct atrophic conditions, hindlimb immobilization and dexamethasone treatment. Leucine supplementation was able to minimize the reduction in rat soleus mass driven by immobilization. On the other hand, leucine supplementation was unable to provide protection against soleus mass loss in dexamethasone treated rats. Interestingly, HMB supplementation was unable to provide protection against mass loss in all treatments. While solely fiber type I cross sectional area (CSA) was protected in immobilized soleus of leucine-supplemented rats, none of the fiber types were protected by leucine supplementation in rats under dexamethasone treatment. In addition and in line with muscle mass results, HMB treatment did not attenuate CSA decrease in all fiber types against either immobilization or dexamethasone treatment. While leucine supplementation was able to minimize increased expression of both Mafbx/Atrogin and MuRF1 in immobilized rats, leucine was only able to minimize Mafbx/Atrogin in dexamethasone treated rats. In contrast, HMB was unable to restrain the increase in those atrogenes in immobilized rats, but in dexamethasone treated rats, HMB minimized increased expression of Mafbx/Atrogin. The amount of ubiquitinated proteins, as expected, was increased in immobilized and dexamethasone treated rats and only leucine was able to block this increase in immobilized rats but not in dexamethasone treated rats. Leucine supplementation maintained soleus tetanic peak force in immobilized rats at normal level. On the other hand, HMB treatment failed to maintain tetanic peak force regardless of treatment. The present data suggested that the anti-atrophic effects of leucine are not mediated by its metabolite HMB.     

New Onset of Constipation during Long-Term Physical Inactivity: A Proof-of-Concept Study on the Immobility-Induced Bowel Changes

Background

The pathophysiological mechanisms underlining constipation are incompletely understood, but prolonged bed rest is commonly considered a relevant determinant.

Aims

Our primary aim was to study the effect of long-term physical inactivity on determining a new onset of constipation. Secondary aim were the evaluation of changes in stool frequency, bowel function and symptoms induced by this prolonged physical inactivity.

Methods

Ten healthy men underwent a 7-day run-in followed by 35-day study of experimentally-controlled bed rest. The study was sponsored by the Italian Space Agency. The onset of constipation was evaluated according to Rome III criteria for functional constipation. Abdominal bloating, flatulence, pain and urgency were assessed by a 100mm Visual Analog Scales and bowel function by adjectival scales (Bristol Stool Form Scale, ease of passage of stool and sense of incomplete evacuation). Daily measurements of bowel movements was summarized on a weekly score. Pre and post bed rest Quality of Life (SF-36), general health (Goldberg’s General Health) and depression mood (Zung scale) questionnaires were administered.

Results

New onset of functional constipation fulfilling Rome III criteria was found in 60% (6/10) of participants (p=0.03). The score of flatulence significantly increased whilst the stool frequency significantly decreased during the week-by-week comparisons period (repeated-measures ANOVA, p=0.02 and p=0.001, respectively). Stool consistency and bowel symptoms were not influenced by prolonged physical inactivity. In addition, no significant changes were observed in general health, in mood state and in quality of life at the end of bed rest

Conclusions

Our results provide evidence that prolonged physical inactivity is relevant etiology in functional constipation in healthy individuals. The common clinical suggestion of early mobilization in bedridden patients is supported as well.