Several complex processes are involved in the production of viable eggs. The aim of this review is to provide an overview on the role played by lysosomal enzymes, especially cathepsins B, D, and L, ...during ovarian follicle growth and maturation. Specific attention is focused on the relationship between the second proteolytic cleavage of yolk proteins (YP) and the resumption of the meiosis during germinal vesicle break down (GVBD). Maturation represents the final stage of oocytes development prior to ovulation. Oocytes in this phase appear translucent. In many teleosts GVBD is accompanied by water uptake and among marine teleosts with pelagic eggs, most of the final volume is reached by this process. The last phase of maturation in benthonic eggs also occurs concomitant to a second proteolytic cleavage and is related with a slight hydration process. In vitro maturation by 17α,20β-dihydroxy-4-pregnen-3one in class III
Danio rerio oocytes, induced 80% of GVBD. The maturation of these oocytes is known to be associated with proteolysis of their major yolk components. In the present study, we show that inhibition of specific enzymes (cathepsins) involved in the second YP processing, did not affect the occurrence of GVBD as the oocytes become translucent and display a slight increase in size. More specifically, in vitro incubation of the maturing oocytes with a cathepsin B inhibitor suppressed both cathepsin B and L activities and the proteolysis of YP. On the contrary, the addition of cathepsin L inhibitor, only affected cathepsin L activity, indicating that cathepsin B is probably involved in Cathepsin L activation, and this enzyme is probably responsible for the second YP processing. These results, together with previous studies, indicate that the GVBD process is independent of the occurrence of the second proteolytic process. It supports the hypothesis that the maturation process is under K
+ ion flux control, while yolk proteolysis is related to the temporal and specific activation of cathepsins by acidification of yolk spheres.
In the present study, a cDNA for the hatching enzyme of a marine tropical fish, Chysiptera parasema, was cloned. This is the first demonstration of hatching enzyme cDNA from a marine tropical fish. ...The amino acid (aa) sequence deduced from the cDNA consisted of an 18-aa signal sequence, a 53-aa propeptide sequence and a 196-aa mature enzyme portion, having a consensus active site sequence for astacin family proteases. Phylogenetic analysis showed that the C. parasema enzyme was included in the clade of HCEs (high choriolytic enzymes), one of the hatching enzymes of freshwater fishes such as medaka (Oryzias latipes), masu salmon (Oncorhynchus masou) and zebrafish (Danio rerio), but not in the group of LCEs (low choriolytic enzymes), another type of hatching enzymes identified in the medaka. The developmental expression patterns of the C. parasema HCE gene were highly similar to that of the medaka HCE gene. The results suggested that the hatching enzyme system is highly conserved between these marine and freshwater fish species. PUBLICATION ABSTRACT
Congenital hypopituitarism (CH) disorders are phenotypically variable. Variants in multiple genes are associated with these disorders, with variable penetrance and inheritance.
We screened a large ...cohort (N = 1765) of patients with or at risk of CH using Sanger sequencing, selected according to phenotype, and conducted next-generation sequencing (NGS) in 51 families within our cohort. We report the clinical, hormonal, and neuroradiological phenotypes of patients with variants in known genes associated with CH.
We identified variants in 178 patients: GH1/GHRHR (51 patients of 414 screened), PROP1 (17 of 253), POU1F1 (15 of 139), SOX2 (13 of 59), GLI2 (7 of 106), LHX3/LHX4 (8 of 110), HESX1 (8 of 724), SOX3 (9 of 354), OTX2 (5 of 59), SHH (2 of 64), and TCF7L1, KAL1, FGFR1, and FGF8 (2 of 585, respectively). NGS identified 26 novel variants in 35 patients (from 24 families). Magnetic resonance imaging showed prevalent hypothalamo-pituitary abnormalities, present in all patients with PROP1, GLI2, SOX3, HESX1, OTX2, LHX3, and LHX4 variants. Normal hypothalamo-pituitary anatomy was reported in 24 of 121, predominantly those with GH1, GHRHR, POU1F1, and SOX2 variants.
We identified variants in 10% (178 of 1765) of our CH cohort. NGS has revolutionized variant identification, and careful phenotypic patient characterization has improved our understanding of CH. We have constructed a flow chart to guide genetic analysis in these patients, which will evolve upon novel gene discoveries.
Abstract
Cyclic peptides are poised to target historically difficult to drug intracellular protein–protein interactions, however, their general cell impermeability poses a challenge for ...characterizing function. Recent advances in microfluidics have enabled permeabilization of the cytoplasmic membrane by physical cell deformation (i.e., mechanoporation), resulting in intracellular delivery of impermeable macromolecules in vector‐ and electrophoretic‐free approaches. However, the number of payloads (e.g., peptides) and/or concentrations delivered via microfluidic mechanoporation is limited by having to pre‐mix cells and payloads, a manually intensive process. In this work, we show that cells are momentarily permeable (
t
1/2
= 1.1–2.8 min) after microfluidic vortex shedding (μVS) and that lower molecular weight macromolecules can be cytosolically delivered upon immediate exposure after cells are processed/permeabilized. To increase the ability to screen peptides, we built a system, dispensing‐microfluidic vortex shedding (DμVS), that integrates a μVS chip with inline microplate‐based dispensing. To do so, we synced an electronic pressure regulator, flow sensor, on/off dispense valve, and an x‐y motion platform in a software‐driven feedback loop. Using this system, we were able to deliver low microliter‐scale volumes of transiently mechanoporated cells to hundreds of wells on microtiter plates in just several minutes (e.g., 96‐well plate filled in <2.5 min). We validated the delivery of an impermeable peptide directed at MDM2, a negative regulator of the tumor suppressor p53, using a click chemistry‐ and NanoBRET‐based cell permeability assay in 96‐well format, with robust delivery across the full plate. Furthermore, we demonstrated that DμVS could be used to identify functional, low micromolar, cellular activity of otherwise cell‐inactive MDM2‐binding peptides using a p53 reporter cell assay in 96‐ and 384‐well format. Overall, DμVS can be combined with downstream cell assays to investigate intracellular target engagement in a high‐throughput manner, both for improving structure–activity relationship efforts and for early proof‐of‐biology of non‐optimized peptide (or potentially other macromolecular) tools.
