CRISPR Typing and Subtyping for Improved Laboratory Surveillance of Salmonella Infections

Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats) is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool

Stroke Rehabilitation Reaches a Threshold

Motor training with the upper limb affected by stroke partially reverses the loss of cortical representation after lesion and has been proposed to increase spontaneous arm use. Moreover, repeated attempts to use the affected hand in daily activities create a form of practice that can potentially lead to further improvement in motor performance. We thus hypothesized that if motor retraining after stroke increases spontaneous arm use sufficiently, then the patient will enter a virtuous circle in which spontaneous arm use and motor performance reinforce each other. In contrast, if the dose of therapy is not sufficient to bring spontaneous use above threshold, then performance will not increase and the patient will further develop compensatory strategies with the less affected hand. To refine this hypothesis, we developed a computational model of bilateral hand use in arm reaching to study the interactions between adaptive decision making and motor relearning after motor cortex lesion. The model contains a left and a right motor cortex, each controlling the opposite arm, and a single action choice module. The action choice module learns, via reinforcement learning, the value of using each arm for reaching in specific directions. Each motor cortex uses a neural population code to specify the initial direction along which the contralateral hand moves towards a target. The motor cortex learns to minimize directional errors and to maximize neuronal activity for each movement. The derived learning rule accounts for the reversal of the loss of cortical representation after rehabilitation and the increase of this loss after stroke with insufficient rehabilitation. Further, our model exhibits nonlinear and bistable behavior: if natural recovery, motor training, or both, brings performance above a certain threshold, then training can be stopped, as the repeated spontaneous arm use provides a form of motor learning that further bootstraps performance and spontaneous use. Below this threshold, motor training is “in vain”: there is little spontaneous arm use after training, the model exhibits learned nonuse, and compensatory movements with the less affected hand are reinforced. By exploring the nonlinear dynamics of stroke recovery using a biologically plausible neural model that accounts for reversal of the loss of motor cortex representation following rehabilitation or the lack thereof, respectively, we can explain previously hard to reconcile data on spontaneous arm use in stroke recovery. Further, our threshold prediction could be tested with an adaptive train–wait–train paradigm: if spontaneous arm use has increased in the “wait” period, then the threshold has been reached, and rehabilitation can be stopped. If spontaneous arm use is still low or has decreased, then another bout of rehabilitation is to be provided

Sex Reversal in Zebrafish fancl Mutants Is Caused by Tp53-Mediated Germ Cell Apoptosis

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA–repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination

3, 3′5 Triiodo L Thyronine Induces Apoptosis in Human Breast Cancer MCF-7cells, Repressing SMP30 Expression through Negative Thyroid Response Elements

Thyroid hormones regulate cell proliferation, differentiation as well as apoptosis. However molecular mechanism underlying apoptosis as a result of thyroid hormone signaling is poorly understood. The antiapoptotic role of Senescence Marker Protein-30 (SMP30) has been characterized in response to varieties of stimuli as well as in knock out model. Our earlier data suggest that thyroid hormone 3, 3'5 Triiodo L Thyronine (T(3)), represses SMP30 in rat liver.In highly metastatic MCF-7, human breast cancer cell line T3 treatment repressed SMP30 expression leading to enhanced apoptosis. Analysis by flow cytometry and other techniques revealed that overexpression and silencing of SMP30 in MCF-7 resulted in decelerated and accelerated apoptosis respectively. In order to identify the cis-acting elements involved in this regulation, we have analyzed hormone responsiveness of transiently transfected hSMP30 promoter deletion reporter vectors in MCF-7 cells. As opposed to the expected epigenetic outcome, thyroid hormone down regulated hSMP30 promoter activity despite enhanced recruitment of acetylated H3 on thyroid response elements (TREs). From the stand point of established epigenetic concept we have categorised these two TREs as negative response elements. Our attempt of siRNA mediated silencing of TRβ, reduced the fold of repression of SMP30 gene expression. In presence of thyroid hormone, Trichostatin- A (TSA), which is a Histone deacetylase (HDAC) inhibitor further inhibited SMP30 promoter activity. The above findings are in support of categorisation of both the thyroid response element as negative response elements as usually TSA should have reversed the repressions.This is the first report of novel mechanistic insights into the remarkable downregulation of SMP30 gene expression by thyroid hormone which in turn induces apoptosis in MCF-7 human breast cancer cells. We believe that our study represents a good ground for future effort to develop new therapeutic approaches to challenge the progression of breast cancer

Antiretroviral-naive and -treated HIV-1 patients can harbour more resistant viruses in CSF than in plasma

Objectives The neurological disorders in HIV-1-infected patients remain prevalent. The HIV-1 resistance in plasma and CSF was compared in patients with neurological disorders in a multicentre study. Methods Blood and CSF samples were collected at time of neurological disorders for 244 patients. The viral loads were >50 copies/mL in both compartments and bulk genotypic tests were realized. Results On 244 patients, 89 and 155 were antiretroviral (ARV) naive and ARV treated, respectively. In ARV-naive patients, detection of mutations in CSF and not in plasma were reported for the reverse transcriptase (RT) gene in 2/89 patients (2.2%) and for the protease gene in 1/89 patients (1.1%). In ARV-treated patients, 19/152 (12.5%) patients had HIV-1 mutations only in the CSF for the RT gene and 30/151 (19.8%) for the protease gene. Two mutations appeared statistically more prevalent in the CSF than in plasma: M41L (P = 0.0455) and T215Y (P = 0.0455). Conclusions In most cases, resistance mutations were present and similar in both studied compartments. However, in 3.4% of ARV-naive and 8.8% of ARV-treated patients, the virus was more resistant in CSF than in plasma. These results support the need for genotypic resistance testing when lumbar puncture is performe