Nuclear RNA-seq of single neurons reveals molecular signatures of activation.

Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully recapitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc and Egr1. SnRNA-seq of mouse dentate granule cells reveals large-scale changes in the activated neuronal transcriptome after brief novel environment exposure, including induction of MAPK pathway genes. In addition, we observe a continuum of activation states, revealing a pseudotemporal pattern of activation from gene expression alone. In summary, snRNA-seq of activated neurons enables the examination of gene expression beyond IEGs, allowing for novel insights into neuronal activation patterns in vivo.

A study on FoxP3 and Tregs in paired samples of peripheral blood and synovium in rheumatoid arthritis.

There is an increasing evidence suggesting the role of fork head boxP3 (FoxP3) in the development and the regulation of CD4(+)CD25(+) Treg cells. T-cell regulatory mechanisms in rheumatoid arthritis patients were evaluated by the contributing factors such as pro-inflammatory cytokines, circulating immune complexes, HLA DR expression, ligand binding biomarkers, FoxP3 expression in paired samples of peripheral blood (PB) and synovial fluid (SF). These cellular responses were further correlated with the humoral immune responses such as anti-cyclic citrullinated peptides IgG (CCP), circulating immune complex-c1q IgG (CIC), immunoglobulin G (IgG) and immunoglobulin M (IgM) of the rheumatoid arthritis factor (RAF). The results suggest a definitive role of Tregs in the homeostatic control because there is an increase in FoxP3 (37%) and HLA-DR (45%) expression in the synovial fluid as compared to PB. Furthermore, humoral responses as a downstream effector mechanism are positively correlated with the pathogenesis of rheumatoid arthritis (RA). A positive relationship exists between quantitative anti-CCP production and the expression of HLA-DR. The study relates an increased and pivotal role of B cell activation in the synovial fluid thereby permitting the need to ablate the targeted B cell immune responses.

Fate and plasticity of the epidermis in response to congenital activation of BRAF.

Germline and somatic mutations in RAS and its downstream effectors are found in several congenital conditions affecting the skin. Here we demonstrate that activation of BRAF in the embryonic mouse ectoderm triggers both craniofacial and skin defects, including hyperproliferation, loss of spinous and granular keratinocyte differentiation, and cleft palate. RNA sequencing of the epidermis confirmed these findings but unexpectedly revealed evidence of continued epidermal maturation, expression of >80% of epidermal differentiation complex genes, and formation of a hydrophobic barrier. Spinous and granular differentiation were restored by pharmacologic inhibition of MAPK/ERK kinase or BRAF. However, restoration of epidermal differentiation was non-cell autonomous and required dermal tissue to be present in tissue recombination studies. These studies indicate that early activation of the RAF signaling pathway in the ectoderm has effects on specific steps of epidermal differentiation, which may be amenable to treatment with currently available pharmacologic inhibitors.

Activated Kras alters epidermal homeostasis of mouse skin, resulting in redundant skin and defective hair cycling.

Germline mutations in the RAS-mitogen-activated protein kinase (RAS/MAPK) pathway are associated with genodermatoses, characterized by cutaneous, cardiac, and craniofacial defects, and cancer predisposition. Whereas activating mutations in HRAS are associated with the vast majority of patients with Costello syndrome, mutations in its paralog, KRAS, are rare. To better understand the disparity among RAS paralogs in human syndromes, we generated mice that activate a gain-of-function Kras allele (Lox-Stop-Lox (LSL)-Kras(G12D)) in ectodermal tissue using two different Cre transgenic lines. Using Msx2-Cre or ligand-inducible keratin 15 (K15)-CrePR, the embryonic effects of activated Kras were bypassed and the effects of Kras(G12D) expression from its endogenous promoter were determined. We found that Kras(G12D) induced redundant skin, papillomas, shortened nails, and hair loss. Redundant skin was associated with basal keratinocyte hyperplasia and an increase in body surface area. Paradoxically, Kras(G12D) also prevented hair cycle activation. We find that Kras(G12D) blocks proliferation in the bulge region of the hair follicle, when activated through Msx2-Cre but not through K15-CrePR. These studies reveal that KRAS, although infrequently involved in RAS/MAPK syndromes, is capable of inducing multiple cutaneous features that grossly resemble human RAS/MAPK syndromes.

Leptospirosis in Madras--a clinical and serological study.

Leptospirosis was confirmed by Microscopic Agglutination Test (MAT) and/or ELISA in 57 patients admitted to the Government General Hospital, Madras, India, during November and December of 1990 and 1991 with symptomatology suggestive of the disease. Fifty (88%) of the 57 cases were males; the mean age of all the cases was 39.6 years (range 17-72). The main clinical features were: fever 100% jaundice 84%, Myalgia 82%, acute renal failure 72% and conjunctival suffusion 58%. Non-azotemic jaundice occurred in 19% of cases. Renal failure was non-oliguric in 24% of cases. 3.5% of patients died. 23 patients underwent peritoneal and/or hemodialysis. ELISA IgM titres ranged from 1:80 to 1:10240 (geometric mean tire 911). MAT titres > or = 1:1600 and > or = 1:800 occurred in 39 of 54 and 51 of 54 cases respectively. Autumnalis was the serogroup most commonly recorded serologically, and Leptospira interrogans serovar autumnalis was isolated from one patient. This study shows that leptospirosis is a significant health problem in Madras, though normally grossly underestimated due to the absence of routine laboratory diagnostic facilities for the disease. Gross under-reporting is also likely in other high rainfall third world areas.