Rational Engineering of an Improved Genetically Encoded pH Sensor Based on Superecliptic pHluorin.

Genetically encoded pH sensors based on fluorescent proteins are valuable tools for the imaging of cellular events that are associated with pH changes, such as exocytosis and endocytosis. Superecliptic pHluorin (SEP) is a pH-sensitive green fluorescent protein (GFP) variant widely used for such applications. Here, we report the rational design, development, structure, and applications of Lime, an improved SEP variant with higher fluorescence brightness and greater pH sensitivity. The X-ray crystal structure of Lime supports the mechanistic rationale that guided the introduction of beneficial mutations. Lime provides substantial improvements relative to SEP for imaging of endocytosis and exocytosis. Furthermore, Lime and its variants are advantageous for a broader range of applications including the detection of synaptic release and neuronal voltage changes.

The presynaptic glycine transporter GlyT2 is regulated by the Hedgehog pathway in vitro and in vivo.

The identity of a glycinergic synapse is maintained presynaptically by the activity of a surface glycine transporter, GlyT2, which recaptures glycine back to presynaptic terminals to preserve vesicular glycine content. GlyT2 loss-of-function mutations cause Hyperekplexia, a rare neurological disease in which loss of glycinergic neurotransmission causes generalized stiffness and strong motor alterations. However, the molecular underpinnings controlling GlyT2 activity remain poorly understood. In this work, we identify the Hedgehog pathway as a robust controller of GlyT2 expression and transport activity. Modulating the activation state of the Hedgehog pathway in vitro in rodent primary spinal cord neurons or in vivo in zebrafish embryos induced a selective control in GlyT2 expression, regulating GlyT2 transport activity. Our results indicate that activation of Hedgehog reduces GlyT2 expression by increasing its ubiquitination and degradation. This work describes a new molecular link between the Hedgehog signaling pathway and presynaptic glycine availability.

Understanding intellectual disability and autism spectrum disorders from common mouse models: synapses to behaviour.

Normal brain development is highly dependent on the timely coordinated actions of genetic and environmental processes, and an aberration can lead to neurodevelopmental disorders (NDDs). Intellectual disability (ID) and autism spectrum disorders (ASDs) are a group of co-occurring NDDs that affect between 3% and 5% of the world population, thus presenting a great challenge to society. This problem calls for the need to understand the pathobiology of these disorders and to design new therapeutic strategies. One approach towards this has been the development of multiple analogous mouse models. This review discusses studies conducted in the mouse models of five major monogenic causes of ID and ASDs: Fmr1, Syngap1, Mecp2, Shank2/3 and Neuroligins/Neurnexins. These studies reveal that, despite having a diverse molecular origin, the effects of these mutations converge onto similar or related aetiological pathways, consequently giving rise to the typical phenotype of cognitive, social and emotional deficits that are characteristic of ID and ASDs. This convergence, therefore, highlights common pathological nodes that can be targeted for therapy. Other than conventional therapeutic strategies such as non-pharmacological corrective methods and symptomatic alleviation, multiple studies in mouse models have successfully proved the possibility of pharmacological and genetic therapy enabling functional recovery.

Primary subcutaneous mixed-type liposarcoma of the thigh showing three simultaneous dedifferentiations: report of an unusual case.

Liposarcoma most commonly occurs in deep soft tissues of the limb and the retroperitoneum; however, its occurrence in the subcutaneous tissue is rare. Mixed-type liposarcoma (MTL) is the rarest subtype. We herein describe a unique case of MTL with three simultaneous dedifferentiations occurring in subcutaneous tissue of left thigh in a 45-year-old male. We briefly review the pertinent literature and report this case because of extreme rarity.

Is rheumatoid factor still a superior test for the diagnosis of rheumatoid arthritis?

The diagnosis of rheumatoid arthritis (RA) is based primarily on the 1987 revised American College of Rheumatology criteria for RA, which considers mainly the clinical symptoms. But typical clinical symptoms of RA are not manifested completely in early disease course. On the other hand, appreciable advantages have been made in the therapeutic strategy of RA in the last decade and highly effective disease-modifying anti-rheumatic drugs are available now for the control of RA. The treatment strategy for the control of early RA is aggressive. Thus, a highly specific and early diagnostic marker is needed for the detection of RA. Our study is an attempt to see the role of anti-CCP2 antibody (claimed to be highly specific and early diagnostic tool) in the diagnosis of RA. We studied 119 cases of RA in terms of clinical symptoms, disease duration and various autoantibody [including rheumatoid factor (RF), anti-CCP2 antibody, antinuclear antibody, anti-dsDNA] and C-reactive protein status. All the tests were also performed in 26 age and sex-matched healthy controls. Estimation of antibodies was done by quantitative ELISA. IgM RF was positive in 47.89% cases (p value = 0.000), followed by IgG RF (42.01%, p = 0.000) and IgA RF (36.97%, p = 0.000). RF was positive in 64.7% RA cases (p value = 0.000) when all three isotypes were tested together. RF was also detected in one healthy control. In 92 cases, anti-CCP2 Ab was done, hence other data were analyzed further in 92 cases only. Anti-CCP2 Ab was positive (cut-off = 15.0 U/ml) in only 50% RA patients but none of the healthy controls was positive for it. Swelling of joints was seen in 82.6% anti-CCP2 Ab positive cases (p value = 0.092) when compared with anti-CCP2 Ab negative cases (67.4%) while among RF positive cases, only 65.4% ((p value = 0.010) cases had swelling of joints. Out of 39 RA cases presenting with disease duration less than 1 year, only 48.71% patients were anti-CCP2 Ab positive while RF was positive in 61.53% patients. Utility of various combined autoantibody tests revealed that if one does all isotypes of RF (IgG, IgA and IgM) only, then 64.7% RA cases can be diagnosed and if anti-CCP2 Ab is added to it, the sensitivity increases to 75.56%. Thus, our study concludes that anti-CCP2 Ab is not a sensitive test for the diagnosis of RA neither it is useful in early diagnosis of RA, but it increases the sensitivity if added with all RF isotypes.