Macaque monkeys show reversed ocular following responses to two-frame-motion stimulus presented with inter-stimulus intervals

Journal of Computational Neuroscience - When two-frame apparent motion stimuli are presented with an appropriate inter-stimulus interval (ISI), motion is perceived in the direction opposite to the...     

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T₀ plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T₁ generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T₀ plants, we could remove foreign DNA easily by genetic segregation in the T₁ generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

     

Comparative analysis of circulating hemocytes of the freshwater snail Pomacea canaliculata

Molluscs are invertebrates of great relevance for economy, environment and public health. The numerous studies on molluscan immunity and physiology registered an impressive variability of circulating hemocytes. This study is focused on the first characterization of the circulating hemocytes of the freshwater gastropod Pomacea canaliculata, a model for several eco-toxicological and parasitological researches.Flow cytometry analysis identified two populations of hemocytes on the basis of differences in size and internal organization. The first population contains small and agranular cells. The second one displays major size and a more articulated internal organization. Light microscopy evidenced two principal morphologies, categorized as Group I (small) and II (large) hemocytes. Group I hemocytes present the characteristics of blast-like cells, with an agranular and basophilic cytoplasm. Group I hemocytes can adhere onto a glass surface but seem unable to phagocytize heat-inactivated Escherichia coli. The majority of Group II hemocytes displays an agranular cytoplasm, while a minority presents numerous granules. Agranular cytoplasm may be basophilic or acidophilic. Granules are positive to neutral red staining and therefore acidic. Independently from their morphology, Group II hemocytes are able to adhere and to engulf heat-inactivated E. coli. Transmission electron microscopy analysis clearly distinguished between agranular and granular hemocytes and highlighted the electron dense content of the granules. After hemolymph collection, time-course analysis indicated that the Group II hemocytes are subjected to an evident dynamism with changes in the percentage of agranular and granular hemocytes. The ability of circulating hemocytes to quickly modify their morphology and stainability suggests that P. canaliculata is endowed with highly dynamic hemocyte populations able to cope with rapid environmental changes as well as fast growing pathogens.     

A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon’s fibroblasts

Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon’s fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2–3 cm² undamaged Tenon’s biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon’s fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon’s and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes.     

Chronic rapamycin treatment or lack of S6K1 does not reduce ribosome activity in vivo

Reducing activity of the mTORC1/S6K1 pathway has been shown to extend lifespan in both vertebrate and invertebrate models. For instance, both pharmacological inhibition of mTORC1 with the drug rapamycin or S6K1 knockout extends lifespan in mice. Since studies with invertebrate models suggest that reducing translational activity can increase lifespan, we reasoned that the benefits of decreased mTORC1 or S6K1 activity might be due, at least in part, to a reduction of general translational activity. Here, we report that mice given a single dose of rapamycin have reduced translational activity, while mice receiving multiple injections of rapamycin over 4 weeks show no difference in translational activity compared with vehicle-injected controls. Furthermore, mice lacking S6K1 have no difference in global translational activity compared with wild-type littermates as measured by the percentage of ribosomes that are active in multiple tissues. Translational activity is reduced in S6K1-knockout mice following single injection of rapamycin, demonstrating that rapamycin’s effects on translation can occur independently of S6K1. Taken together, these data suggest that benefits of chronic rapamycin treatment or lack of S6K1 are dissociable from potential benefits of reduced translational activity, instead pointing to a model whereby changes in translation of specific subsets of mRNAs and/or translation-independent effects of reduced mTOR signaling underlie the longevity benefits.     

Increased Renal Iron Accumulation in Hypertensive Nephropathy of Salt-Loaded Hypertensive Rats

Although iron is reported to be associated with the pathogenesis of chronic kidney disease, it is unknown whether iron participates in the pathophysiology of nephrosclerosis. Here, we investigate whether iron is involved in the development of hypertensive nephropathy and the effects of iron restriction on nephrosclerosis in salt- loaded stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP were given either a normal or high-salt diet for 8 weeks. Another subset of SHRSP were fed a high-salt with iron-restricted diet. SHRSP given a high-salt diet developed severe hypertension and nephrosclerosis. As a result, survival rate was decreased after 8 weeks diet. Importantly, massive iron accumulation and increased iron content were observed in the kidneys of salt-loaded SHRSP, along with increased superoxide production, urinary 8-Hydroxy-2′-deoxyguanosine excretion, and urinary iron excretion; however, these changes were markedly attenuated by iron restriction. Of interest, expression of cellular iron transport proteins, transferrin receptor 1 and divalent metal transporter 1, was increased in the tubules of salt-loaded SHRSP. Notably, iron restriction attenuated the development of severe hypertension and nephrosclerosis, thereby improving survival rate in salt-loaded SHRSP. Taken together, these results suggest a novel mechanism by which iron plays a role in the development of hypertensive nephropathy and establish the effects of iron restriction on salt-induced nephrosclerosis.     

Dam Methylation Participates in the Regulation of PmrA/PmrB and RcsC/RcsD/RcsB Two Component Regulatory Systems in Salmonella enterica Serovar Enteritidis

The absence of Dam in Salmonella enterica serovar Enteritidis causes a defect in lipopolysaccharide (LPS) pattern associated to a reduced expression of wzz gene. Wzz is the chain length regulator of the LPS O-antigen. Here we investigated whether Dam regulates wzz gene expression through its two known regulators, PmrA and RcsB. Thus, the expression of rcsB and pmrA was monitored by quantitative real-time RT-PCR and Western blotting using fusions with 3×FLAG tag in wild type (wt) and dam strains of S. Enteritidis. Dam regulated the expression of both rcsB and pmrA genes; nevertheless, the defect in LPS pattern was only related to a diminished expression of RcsB. Interestingly, regulation of wzz in serovar Enteritidis differed from that reported earlier for serovar Typhimurium; RcsB induces wzz expression in both serovars, whereas PmrA induces wzz in S. Typhimurium but represses it in serovar Enteritidis. Moreover, we found that in S. Enteritidis there is an interaction between both wzz regulators: RcsB stimulates the expression of pmrA and PmrA represses the expression of rcsB. Our results would be an example of differential regulation of orthologous genes expression, providing differences in phenotypic traits between closely related bacterial serovars.     

Therapeutic Efficacy of C-Kit-Targeted Radioimmunotherapy Using 90Y-Labeled Anti-C-Kit Antibodies in a Mouse Model of Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive tumor and prognosis remains poor. Therefore, the development of more effective therapy is needed. We previously reported that high levels of an anti-c-kit antibody (12A8) accumulated in SCLC xenografts. In the present study, we evaluated the efficacy of two antibodies (12A8 and 67A2) for radioimmunotherapy (RIT) of an SCLC mouse model by labeling with the ⁹⁰Y isotope.

Methods

¹¹¹In- or ¹²⁵I-labeled antibodies were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays in c-kit-expressing SY cells and in vivo by biodistribution in SY-bearing mice. Therapeutic efficacy of ⁹⁰Y-labeled antibodies was evaluated in SY-bearing mice upto day 28 and histological analysis was conducted at day 7.

Results

[¹¹¹In]12A8 and [¹¹¹In]67A2 specifically bound to SY cells with high affinity (8.0 and 1.9 nM, respectively). 67A2 was internalized similar to 12A8. High levels of [¹¹¹In]12A8 and [¹¹¹In]67A2 accumulated in tumors, but not in major organs. [¹¹¹In]67A2 uptake by the tumor was 1.7 times higher than for [¹¹¹In]12A8. [⁹⁰Y]12A8, but not [⁹⁰Y]67A2, suppressed tumor growth in a dose-dependent manner. Tumors treated with 3.7 MBq of [⁹⁰Y]12A8, and 1.85 and 3.7 MBq of [⁹⁰Y]67A2 (absorbed doses were 21.0, 18.0 and 35.9 Gy, respectively) almost completely disappeared approximately 2 weeks after injection, and regrowth was not observed except for in one mouse treated with 1.85 MBq [⁹⁰Y]67A2. The area of necrosis and fibrosis increased depending on the RIT effect. Apoptotic cell numbers increased with increased doses of [⁹⁰Y]12A8, whereas no dose-dependent increase was observed following [⁹⁰Y]67A2 treatment. Body weight was temporarily reduced but all mice tolerated the RIT experiments well.

Conclusion

Treatment with [⁹⁰Y]12A8 and [⁹⁰Y]67A2 achieved a complete therapeutic response when SY tumors received an absorbed dose greater than 18 Gy and thus are promising RIT agents for metastatic SCLC cells at distant sites.