A microfluidic platform towards automated multiplexed in situ sequencing.

Advancements in multiplexed in situ RNA profiling techniques have given unprecedented insight into spatial organization of tissues by enabling single-molecule quantification and sub-micron localization of dozens to thousands of RNA species simultaneously in cells and entire tissue sections. However, the lack of automation of the associated complex experimental procedures represents a potential hurdle towards their routine use in laboratories. Here, we demonstrate an approach towards automated generation and sequencing of barcoded mRNA amplicons in situ, directly in fixed cells. This is achieved through adaptation of a microfluidic tool compatible with standard microscope slides and cover glasses. The adapted tool combines a programmable reagent delivery system with temperature controller and flow cell to perform established in situ sequencing protocols, comprising hybridization and ligation of gene-specific padlock probes, rolling circle amplification of the probes yielding barcoded amplicons and identification of amplicons through barcode sequencing. By adapting assay parameters (e.g. enzyme concentration and temperature), we achieve a near-identical performance in identifying mouse beta-actin transcripts, in comparison with the conventional manual protocol. The technically adapted assay features i) higher detection efficiency, ii) shorter protocol time, iii) lower consumption of oligonucleotide reagents but slightly more enzyme. Such an automated microfluidic tissue processor for in situ sequencing studies would greatly enhance its research potentials especially for cancer diagnostics, thus paving way to rapid and effective therapies.

[Italian multicenter study for the verification of the efficacy and tolerability of short-term substitution hormone therapy using conjugated estrogens and progestagens administered orally in the postmenopausal a period]. / Studio multicentrico italiano di verifica in merito all'efficacia ed alla tollerabilità della terapia ormonale sostitutiva a breve termine con estrogeni coniugati e progestinici per via orale nella postmenopausa.

Three hundred and sixty-three postmenopausal women received 6 months of cyclic hormone replacement therapy with conjugated estrogens in differing doses (0.625 mg and 1.25 mg) associated in a sequential pattern lasting 12 days with progestagens (medroxyprogesterone acetate or medrogestone) in 16 italian health centres. The results of the multicentre study confirm the efficacy of this conjugated estrogen-progestogen regimen in resolving climacteric syndrome, offering cardiovascular protection and vaginal trophic effect and preventing postmenopausal osteoporosis, as well as confirming its tolerability in relation to blood pressure, body weight, breast, endometrium, blood coagulation and hepato-renal function.