NC1-Peptide From Collagen α3 (IV) Chains in the Basement Membrane of Testes Regulates Spermatogenesis via p-FAK-Y407

Abstract The blood–testis barrier (BTB) in the testis is an important ultrastructure to support spermatogenesis. This blood-tissue barrier undergoes remodeling at late stage VII to early stage IX of the epithelial cycle to support the transport of preleptotene spermatocytes across the BTB to prepare for meiosis I/II at the apical compartment through a mechanism that remains to be delineated. Studies have shown that NC1-peptide-derived collagen α3 (IV) chain in the basement membrane is a bioactive peptide that induces BTB remodeling. It also promotes the release of fully developed spermatids into the tubule lumen. Thus, this endogenously produced peptide coordinates these 2 cellular events across the seminiferous epithelium. Using an NC1-peptide complementary deoxyribonucleic acid (cDNA) construct to transfect adult rat testes for overexpression, NC1-peptide was found to effectively induce germ cell exfoliation and BTB remodeling, which was associated with a surge and activation of p-rpS6, the downstream signaling protein of mTORC1 and the concomitant downregulation of p-FAK-Y407 in the testis. In order to define the functional relationship between p-rpS6 and p-FAK-Y407 signaling to confer the ability of NC1-peptide to regulate testis function, a phosphomimetic (and thus constitutively active) mutant of p-FAK-Y407 (p-FAK-Y407E-MT) was used for its co-transfection, utilizing Sertoli cells cultured in vitro with a functional tight junction (TJ) barrier that mimicked the BTB in vivo. Overexpression of p-FAK-Y407E-MT blocked the effects of NC1-peptide to perturb Sertoli cell BTB function by promoting F-actin and microtubule cytoskeleton function, and downregulated the NC1-peptide-mediated induction of p-rpS6 activation. In brief, NC1-peptide is an important endogenously produced biomolecule that regulates BTB dynamics.

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LINC00680 enhances hepatocellular carcinoma stemness behavior and chemoresistance by sponging miR-568 to upregulate AKT3

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01854-5 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Gege Shu ◽

Huizhao Su ◽

Zhiqian Wang ◽

Shihui Lai ◽

Yan Wang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Xenograft Model ◽

Luciferase Reporter ◽

Loss Of Function ◽

Mechanism Study ◽

Downstream Signaling ◽

Mouse Xenograft Model ◽

High Level

Abstract Background Hepatocellular carcinoma (HCC) has an extremely poor prognosis due to the development of chemoresistance, coupled with inherently increased stemness properties. Long non-coding RNAs (LncRNAs) are key regulators for tumor cell stemness and chemosensitivity. Currently the relevance between LINC00680 and tumor progression was still largely unknown, with only one study showing its significance in glioblastoma. The study herein was aimed at identifying the role of LINC00680 in the regulation HCC stemness and chemosensitivity. Methods QRT-PCR was used to detect the expression of LINC00680, miR-568 and AKT3 in tissue specimen and cell lines. Gain- or loss-of function assays were applied to access the function of LINC00680 in HCC cells, including cell proliferation and stemness properties. HCC stemness and chemosensitivity were determined by sphere formation, cell viability and colony formation. Luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays were performed to examine the interaction between LINC00680 and miR-568 as well as that between miR-568 and AKT3. A nude mouse xenograft model was established for the in vivo study. Results We found that LINC00680 was remarkably upregulated in HCC tissues. Patients with high level of LINC00680 had poorer prognosis. LINC00680 overexpression significantly enhanced HCC cell stemness and decreased in vitro and in vivo chemosensitivity to 5-fluorouracil (5-Fu), whereas LINC00680 knockdown led to opposite results. Mechanism study revealed that LINC00680 regulated HCC stemness and chemosensitivity through sponging miR-568, thereby expediting the expression of AKT3, which further activated its downstream signaling molecules, including mTOR, elF4EBP1, and p70S6K. Conclusion LINC00680 promotes HCC stemness properties and decreases chemosensitivity through sponging miR-568 to activate AKT3, suggesting that LINC00680 might be a potentially important HCC diagnosis marker and therapeutic target.

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A Detailed Examination of the in vivo and in vitro Effects of ACTH on Gonadotropin Secretion in the Adult Rat

Neuroendocrinology ◽

10.1159/000124090 ◽

1985 ◽

Vol 40 (4) ◽

pp. 297-302 ◽

Cited By ~ 18

Author(s):

David R. Mann ◽

Diane Evans ◽

Festus Edoimioya ◽

Freja Kamel ◽

George M. Butterstein

Keyword(s):

Detailed Examination ◽

Gonadotropin Secretion ◽

Adult Rat ◽

In Vitro Effects

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Upregulations of Clcn3 and P-Gp Provoked by Lens Osmotic Expansion in Rat Galactosemic Cataract

Journal of Diabetes Research ◽

10.1155/2017/3472735 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8

Author(s):

Lixia Ji ◽

Lixia Cheng ◽

Zhihong Yang

Keyword(s):

Osmotic Stress ◽

Early Stage ◽

Lens Epithelial Cells ◽

Anterior Capsule ◽

Glycoprotein P ◽

Protein Levels ◽

P Glycoprotein ◽

Sugar Cataract

Objective.Lens osmotic expansion, provoked by overactivated aldose reductase (AR), is the most essential event of sugar cataract. Chloride channel 3 (Clcn3) is a volume-sensitive channel, mainly participating in the regulation of cell fundamental volume, and P-glycoprotein (P-gp) acts as its modulator. We aim to study whether P-gp and Clcn3 are involved in lens osmotic expansion of galactosemic cataract.Methods and Results.In vitro, lens epithelial cells (LECs) were primarily cultured in gradient galactose medium (10–60 mM), more and more vacuoles appeared in LEC cytoplasm, and mRNA and protein levels of AR, P-gp, and Clcn3 were synchronously upregulated along with the increase of galactose concentration. In vivo, we focused on the early stage of rat galactosemic cataract, amount of vacuoles arose from equatorial area and scattered to the whole anterior capsule of lenses from the 3rd day to the 9th day, and mRNA and protein levels of P-gp and Clcn3 reached the peak around the 9th or 12th day.Conclusion. Galactosemia caused the osmotic stress in lenses; it also markedly leads to the upregulations of AR, P-gp, and Clcn3 in LECs, together resulting in obvious osmotic expansion in vitro and in vivo.

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Migration of leukocytes through the vessel wall and beyond

Thrombosis and Haemostasis ◽

10.1160/th03-04-0220 ◽

2003 ◽

Vol 90 (10) ◽

pp. 598-606 ◽

Cited By ~ 56

Author(s):

Rashmi Yadav ◽

Karen Larbi ◽

Rebecca Young ◽

Sussan Nourshargh

Keyword(s):

Basement Membrane ◽

Vessel Wall ◽

International Workshop ◽

Medical Science ◽

Blood Stream ◽

Primary Response ◽

Leukocyte Migration ◽

Leukocyte Transmigration ◽

Cellular Events

SummaryThe migration of leukocytes from the vascular lumen to sites of infection and/or injury in the extravascular tissue involves a series of sequential and coordinated molecular and cellular events with the resultant primary response being that of reduced leukocyte velocity within the blood stream, followed by leukocyte firm adhesion to endothelial cells lining the vessel wall and eventually migration through the vessel wall. Despite the growing knowledge of the mechanisms that mediate initial interaction of leukocytes with the endothelium, very little is known about the mechanisms that mediate and regulate leukocyte migration through the venular wall, the endothelium and its associated perivascular basement membrane. This review, whilst giving a brief outline of the stepwise cascade of molecular interactions involved in this process and the methods employed to investigate leukocyte migration in vivo, focuses primarily on mechanisms of leukocyte transmigration, the final step in the process of leukocyte emigration. Furthermore, special emphasis is placed on discussing the process and the mechanisms involved in leukocyte migration through the basement membrane, a structure that presents significant impedance to transmigrating leukocytes but is seldom investigated in the context of leukocyte transmigration in vivo. The review also discusses the growing evidence supporting the concept that leukocyte transmigration is not only a response that describes the passage of leukocytes through the venular wall, but also acts as a means of regulating leukocyte responsiveness beyond the vessel wall, i.e. within the extravascular tissue.This publication was partially financed by Serono Foundation for the Advancement of Medical Science.Part of this paper was originally presented at the 2nd International Workshop on New Therapeutic Targets in Vascular Biology from February 6-9, 2003 in Geneva, Switzerland.

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Effects of hypophysectomy and subsequent FSH and testosterone treatment on inhibin production by adult rat testes

Journal of Endocrinology ◽

10.1677/joe.0.1050001 ◽

1985 ◽

Vol 105 (1) ◽

pp. 1-6 ◽

Cited By ~ 26

Author(s):

C. L. Au ◽

D. M. Robertson ◽

D. M. de Kretser

Keyword(s):

Seminiferous Tubule ◽

Hormonal Control ◽

Human Chorionic Gonadotrophin ◽

Experimental Conditions ◽

Adult Rats ◽

Adult Rat ◽

Fluid Production ◽

Tubule Fluid

ABSTRACT The hormonal control of inhibin production by adult rat testes was investigated using an in-vitro inhibin bioassay validated for the measurement of inhibin activity in charcoal-treated rat testicular extracts. The effect of hypophysectomy examined at 16 h, 3, 7 and 42 days after surgery showed a decrease in testicular inhibin content and seminiferous tubule fluid production by 7 days and a decrease in inhibin production by 42 days. Serum FSH and LH were suppressed 3 days after surgery. In 30-day chronically hypophysectomized adult rats treated for 3 days with twice daily s.c. injections of (a) human FSH (hFSH, 22 i.u./rat per day), (b) testosterone (5 mg/rat per day), (c) hFSH + testosterone (same doses as a and b), or (d) human chorionic gonadotrophin (hCG, 12 i.u./rat per day), hFSH or hFSH and testosterone stimulated an increase in testicular inhibin content but not in inhibin production or tubule fluid production. Testosterone and hCG had no effect on these parameters. It is concluded that in vivo, FSH alone stimulates an increase in testicular inhibin content. The failure to observe an increase in inhibin production in vivo is attributed to the suppression of seminiferous tubule fluid production under the same experimental conditions. J. Endocr. (1985) 105, 1–6

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Rationally Designing Simple Rod-Like Amphiphilic NIR-Emissive AIE Probes for Precise In-Vivo Detection of Aβ Fibrils/Plaques at A Super-Early Stage

10.26434/chemrxiv.13699222.v1 ◽

2021 ◽

Author(s):

Yipu Wang ◽

Dong Mei ◽

Xinyi Zhang ◽

Da-Hui Qu ◽

Ju Mei ◽

...

Keyword(s):

High Performance ◽

Ex Vivo ◽

Early Stage ◽

Signal To Noise Ratio ◽

High Signal ◽

In Vivo Detection ◽

Different Strains ◽

Aβ Fibrils

With increase of social aging, Alzheimer's disease (AD) has been one of the serious diseases threatening human health. The occurrence of Aβ fibrils or plaques is recognized as the hallmark of AD. Currently, optical imaging has stood out to be a promising technique for the imaging of Aβ fibrils/plaques and the diagnosis of AD. However, restricted by their poor blood-brain barrier (BBB) penetrability, short-wavelength excitation and emission, and aggregation-caused quenching (ACQ) effect, the clinically used gold-standard optical probes such as <a>thioflavin</a> T (ThT) and thioflavin S (ThS), are not effective enough in the early diagnosis of AD in vivo. Herein, we put forward an “all-in-one” design principle and demonstrate its feasibility in developing high-performance fluorescent probes which are specific to Aβ fibrils/plaques and promising for super-early in-vivo diagnosis of AD. As a proof of concept, a simple rod-like amphiphilic NIR fluorescent AIEgen, i.e., AIE-CNPy-AD, is developed by taking the specificity, BBB penetration ability, deep-tissue penetration capacity, high signal-to-noise ratio (SNR) into consideration. AIE-CNPy-AD is constituted by connecting the electron-donating and accepting moieties through single bonds and tagging with a propanesulfonate tail, giving rise to the NIR fluorescence, aggregation-induced emission (AIE) effect, amphiphilicity, and rod-like structure, which in turn result in high binding-affinity and excellent specificity to Aβ fibrils/plaques, satisfactory ability to penetrate BBB and deep tissues, ultrahigh SNR and sensitivity, and high-fidelity imaging capability. In-vitro, ex-vivo, and in-vivo <a>identifying of Aβ fibrils/plaques</a> in different strains of mice indicate that AIE-CNPy-AD holds the universality to the detection of Aβ fibrils/plaques. It is noteworthy that AIE-CNPy-AD is even able to trace the small and sparsely distributed Aβ fibrils/plaques in very young AD model mice such as 4-month-old APP/PS1 mice which are reported to be the youngest mice to have Aβ deposits in brains, suggesting its great potential in diagnosis and intervention of AD at a super-early stage.

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Defining the early stages of intestinal colonisation by whipworms

10.1101/2020.08.21.261586 ◽

2020 ◽

Author(s):

María A. Duque-Correa ◽

David Goulding ◽

Claire Cormie ◽

Catherine Sharpe ◽

Judit Gali Moya ◽

...

Keyword(s):

Intestinal Epithelium ◽

Early Stage ◽

Proximal Colon ◽

Host Cells ◽

Parasitic Nematodes ◽

Metazoan Parasites ◽

Multiple Cells ◽

First Time

ABSTRACTHundreds of millions of people are infected with whipworms (Trichuris trichiura), large metazoan parasites that live in the caecum and proximal colon. Whipworms inhabit distinct multi-intracellular epithelial burrows that have been described as syncytial tunnels. However, the interactions between first-stage (L1) larvae and the host epithelia that determine parasite invasion and establishment in the syncytium remain unclear. In vivo experiments investigating these events have been severely hampered by the limited in situ accessibility to intracellular infective larvae at the bottom of the crypts of Lieberkühn, and the lack of genetic tools such as fluorescent organisms that are readily available for other pathogens but not parasitic nematodes. Moreover, cell lines, which do not mimic the complexity of the intestinal epithelium, have been unsuccessful in supporting infection by whipworm larvae. Here, we show that caecaloids grown in an open crypt-like conformation recapitulate the caecal epithelium. Using this system, we establish in vitro infections with T. muris L1 larvae for the first-time, and provide clear evidence that syncytial tunnels are formed at this early stage. We show that larval whipworms are completely intracellular but woven through multiple cells. Using the caecaloids, we are able to visualise the pathways taken by the larvae as they burrow through the epithelial cells. We also demonstrate that larvae degrade the mucus layers overlaying the epithelium, enabling them to access the cells below. We show that early syncytial tunnels are composed of enterocytes and goblet cells that are alive and actively interacting with the larvae during the first 24 h of the infection. Progression of infection results in damage to host cells and by 72 h post-infection, we show that desmosomes of cells from infected epithelium widen and some host cells appear to become liquified. Collectively, our work unravels processes mediating the intestinal epithelium invasion by whipworms and reveals new specific interactions between the host and the parasite that allow the whipworm to establish on its multi-intracellular niche. Our study demonstrates that caecaloids can be used as a relevant in vitro model to investigate the infection biology of T. muris during the early colonisation of its host.

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TArget-Responsive Subcellular Catabolism Analysis for Early-stage Antibody-Drug Conjugates Screening and Assessment

10.26434/chemrxiv.13309454 ◽

2020 ◽

Author(s):

Hua Sang ◽

Jiali Liu ◽

Fang Zhou ◽

Xiaofang Zhang ◽

Jingwei Zhang ◽

...

Keyword(s):

Quality Assessment ◽

Therapeutic Efficacy ◽

High Throughput Screening ◽

Early Stage ◽

Cellular Level ◽

Drug Conjugates ◽

Therapeutic Outcomes ◽

Lysosomal Proteolysis

Key events including antibody-antigen affinity, ADC internalization, trafficking and lysosomal proteolysis-mediated payload release combinatorially determine the therapeutic efficacy and safety for ADCs. Nevertheless, a universal technology that efficiently and conveniently evaluates the involvement of these above elements to ADC payload release and hence the final therapeutic outcomes for mechanistic studies and quality assessment is lacking. Considering the plethora of ADC candidates under development owing to the ever-evolving linker and drug chemistry, we developed a TArget-Responsive Subcellular Catabolism (TARSC) approach that measures catabolites kinetics for given ADCs and elaborates how each individual step ranging from antigen binding to lysosomal proteolysis affects ADC catabolism by targeted interferences. Using a commercial and a biosimilar ado-trastuzumab emtansine (T-DM1) as model ADCs, we recorded unequivocal catabolites kinetics for the two T-DM1s in the presence and absence of the targeted interferences. Their negligible differences in TARSC profiles fitting with their undifferentiated therapeutic outcomes suggested by in vitro viability assays and in vivo tumor growth assays, highlighting TARSC analysis as a good indicator of ADC efficacy and bioequivalency. Lastly, we demonstrated the use of TARSC in assessing payload release efficiency for a new Trastuzumab-toxin conjugate. Collectively, we demonstrated the use of TARSC in characterizing ADC catabolism at (sub)cellular level, and in systematically depicting whether given target proteins affect ADC payload release and hence therapeutic efficacy. We anticipate its future use in high-throughput screening, quality assessment and mechanistic understanding of ADCs for drug R&D before proceeding to costly in vivo experiments.

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Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos

10.21203/rs.3.rs-73275/v2 ◽

2020 ◽

Author(s):

Vera A van der Weijden ◽

Meret Schmidhauser ◽

Mayuko Kurome ◽

Johannes Knubben ◽

Veronika L Flöter ◽

...

Keyword(s):

Signaling Pathways ◽

Early Stage ◽

Developmental Competence ◽

Molecular Signaling ◽

Developmental Potential ◽

Stage Of Development ◽

Molecular Signaling Pathways ◽

Canonical Pathways

Abstract Background: The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts.Results: In vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation, tRNA charging, and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14-3-3-mediated signaling, signaling of Rho family GTPases, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts.Conclusions: Our findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.

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CSPα reduces aggregates and rescues striatal dopamine release in αsynuclein transgenic mice

10.1101/2020.07.31.229153 ◽

2020 ◽

Author(s):

L Caló ◽

E Hidari ◽

M Wegrzynowicz ◽

JW Dalley ◽

BL Schneider ◽

...

Keyword(s):

Dopamine Release ◽

Early Stage ◽

Synaptic Function ◽

Snare Complex ◽

Early Event ◽

Vesicle Recycling ◽

Cysteine String Protein ◽

And Function

AbstractαSynuclein aggregation at the synapse is an early event in Parkinson’s disease and is associated with impaired striatal synaptic function and dopaminergic neuronal death. The cysteine string protein (CSPα) and αsynuclein have partially overlapping roles in maintaining synaptic function and mutations in each cause neurodegenerative diseases. CSPα is a member of the DNAJ/HSP40 family of co-chaperones and like αsynuclein, chaperones the SNARE complex assembly and neurotransmitter release. αSynuclein can rescue neurodegeneration in CSPαKO mice. However, whether αsynuclein aggregation alters CSPα expression and function is unknown. Here we show that αsynuclein aggregation at the synapse induces a decrease in synaptic CSPα and a reduction in the complexes that CSPα forms with HSC70 and STGa. We further show that viral delivery of CSPα rescues in vitro the impaired vesicle recycling in PC12 cells with αsynuclein aggregates and in vivo reduces synaptic αsynuclein aggregates restoring normal dopamine release in 1-120hαsyn mice. These novel findings reveal a mechanism by which αsynuclein aggregation alters CSPα at the synapse, and show that CSPα rescues αsynuclein aggregation-related phenotype in 1-120hαsyn mice similar to the effect of αsynuclein in CSPαKO mice. These results implicate CSPα as a potential therapeutic target for the treatment of early-stage PD.

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