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Article: De novo

Cummins, Matthew C / Tripathy, Ashutosh / Sondek, John / Kuhlman, Brian

bioRxiv : the preprint server for biology

2023

Abstract: Many protein therapeutics are competitive inhibitors that function by binding to endogenous proteins and preventing them from interacting with native partners. One effective strategy for engineering competitive inhibitors is to graft structural motifs ... ...

Abstract	Many protein therapeutics are competitive inhibitors that function by binding to endogenous proteins and preventing them from interacting with native partners. One effective strategy for engineering competitive inhibitors is to graft structural motifs from a native partner into a host protein. Here, we develop and experimentally test a computational protocol for embedding binding motifs in de novo designed proteins. The protocol uses an "inside-out" approach: Starting with a structural model of the binding motif docked against the target protein, the de novo protein is built by growing new structural elements off the termini of the binding motif. During backbone assembly, a score function favors backbones that introduce new tertiary contacts within the designed protein and do not introduce clashes with the target binding partner. Final sequences are designed and optimized using the molecular modeling program Rosetta. To test our protocol, we designed small helical proteins to inhibit the interaction between Gα Statement for broader audience: Engineered proteins that bind to specific target proteins are useful as research reagents, diagnostics, and therapeutics. We used computational protein design to engineer de novo proteins that bind and competitively inhibit the G protein, Gα
Language	English
Publishing date	2023-03-28
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.03.28.534629
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Article ; Online: De novo design of stable proteins that efficaciously inhibit oncogenic G proteins.

Cummins, Matthew C / Tripathy, Ashutosh / Sondek, John / Kuhlman, Brian

Protein science : a publication of the Protein Society

2023 Volume 32, Issue 8, Page(s) e4713

Abstract	Many protein therapeutics are competitive inhibitors that function by binding to endogenous proteins and preventing them from interacting with native partners. One effective strategy for engineering competitive inhibitors is to graft structural motifs from a native partner into a host protein. Here, we develop and experimentally test a computational protocol for embedding binding motifs in de novo designed proteins. The protocol uses an "inside-out" approach: Starting with a structural model of the binding motif docked against the target protein, the de novo protein is built by growing new structural elements off the termini of the binding motif. During backbone assembly, a score function favors backbones that introduce new tertiary contacts within the designed protein and do not introduce clashes with the target binding partner. Final sequences are designed and optimized using the molecular modeling program Rosetta. To test our protocol, we designed small helical proteins to inhibit the interaction between Gα
MeSH term(s)	Isoenzymes ; Protein Binding ; Models, Molecular ; GTP-Binding Proteins ; Protein Engineering/methods
Chemical Substances	Isoenzymes ; GTP-Binding Proteins (EC 3.6.1.-)
Language	English
Publishing date	2023-06-27
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	1106283-6
ISSN	1469-896X ; 0961-8368
ISSN (online)	1469-896X
ISSN	0961-8368
DOI	10.1002/pro.4713
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Article ; Online: Design of a protease-activated PD-L1 inhibitor.

Goudy, Odessa J / Peng, Alice / Tripathy, Ashutosh / Kuhlman, Brian

Protein science : a publication of the Protein Society

2023 Volume 32, Issue 3, Page(s) e4578

Abstract: Immune checkpoint inhibitors that bind to the cell surface receptor PD-L1 are effective anti-cancer agents but suffer from immune-related adverse events as PD-L1 is expressed on both healthy and cancer cells. To mitigate toxicity, researchers are testing ...

Abstract	Immune checkpoint inhibitors that bind to the cell surface receptor PD-L1 are effective anti-cancer agents but suffer from immune-related adverse events as PD-L1 is expressed on both healthy and cancer cells. To mitigate toxicity, researchers are testing prodrugs that have low affinity for checkpoint targets until activated with proteases enriched in the tumor microenvironment. Here, we engineer a prodrug form of a PD-L1 inhibitor. The inhibitor is a soluble PD-1 mimetic that was previously engineered to have high affinity for PD-L1. In the basal state, the binding surface of the PD-1 mimetic is masked by fusing it to a soluble variant of its natural ligand, PD-L1. Proteolytic cleavage of the linker that connects the mask to the inhibitor activates the molecule. To optimize the mask so that it effectively blocks binding to PD-L1 but releases upon cleavage, we tested a set of mutants with varied affinity for the inhibitor. The top-performing mask reduces the affinity of the prodrug for PD-L1 120-fold, and binding is nearly fully recovered upon cleavage. In a cell-based assay measuring inhibition of the PD-1:PD-L1 interaction on the surface of cells, the IC50s of the masked inhibitors were up to 40-fold higher than their protease-treated counterparts. The changes in activity we observe upon protease treatment are comparable to systems currently tested in the clinic and provide evidence that natural binding partners are an excellent starting point for creating a prodrug.
MeSH term(s)	Immune Checkpoint Inhibitors ; B7-H1 Antigen/metabolism ; Peptide Hydrolases ; Programmed Cell Death 1 Receptor/metabolism ; Prodrugs
Chemical Substances	Immune Checkpoint Inhibitors ; B7-H1 Antigen ; Peptide Hydrolases (EC 3.4.-) ; Programmed Cell Death 1 Receptor ; Prodrugs
Language	English
Publishing date	2023-01-23
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	1106283-6
ISSN	1469-896X ; 0961-8368
ISSN (online)	1469-896X
ISSN	0961-8368
DOI	10.1002/pro.4578
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Article ; Online: Stoichiometry for entry and binding properties of the Env protein of R5 T cell-tropic HIV-1 and its evolutionary variant of macrophage-tropic HIV-1.

Bonner, Xavier / Sondgeroth, Amy / McCue, Amelia / Nicely, Nathan / Tripathy, Ashutosh / Spielvogel, Ean / Moeser, Matthew / Ke, Ruian / Leiderman, Karin / Joseph, Sarah B / Swanstrom, Ronald

mBio

2024 Volume 15, Issue 4, Page(s) e0032124

Abstract: Human immunodeficiency virus type 1 typically requires a high density of CD4 for efficient entry as a mechanism to target CD4+ T cells (T-tropic), with CCR5 being used most often as the coreceptor. When target T cells are limiting, the virus can evolve ... ...

Abstract	Human immunodeficiency virus type 1 typically requires a high density of CD4 for efficient entry as a mechanism to target CD4+ T cells (T-tropic), with CCR5 being used most often as the coreceptor. When target T cells are limiting, the virus can evolve to infect cells with a low density of CD4 such as macrophages (M-tropic). The entry phenotype is known to be encoded in the viral Env protein on the surface of the virus particle. Using data showing a dose response for infectivity based on CD4 surface density, we built a model consistent with T-tropic viruses requiring multiple CD4 molecules to mediate infection, whereas M-tropic viruses can infect cells using a single CD4 receptor molecule interaction. We also found that T-tropic viruses bound to the surface of cells with a low density of CD4 are released more slowly than M-tropic viruses which we modeled to be due to multiple interactions of the T-tropic virus with multiple CD4 molecules to allow the initial stable binding. Finally, we found that some M-tropic Env proteins, as the gp120 subunit, possess an enhanced affinity for CD4 compared with their T-tropic pair, indicating that the evolution of macrophage tropism can be reflected both in the closed Env trimer conformation on the virion surface and, in some cases, also in the open confirmation of gp120 Env. Collectively, these studies reveal differences in the stoichiometry of interaction of T-tropic and M-tropic viruses with CD4 and start to identify the basis of binding differences at the biochemical level. Importance: Human immunodeficiency virus type 1 normally targets CD4+ T cells for viral replication. When T cells are limiting, the virus can evolve to infect myeloid cells. The evolutionary step involves a change from requiring a high surface density of CD4 for entry to being able to infect cells with a low density of CD4, as is found on myeloid lineage cells such as macrophage and microglia. Viruses able to infect macrophages efficiently are most often found in the CNS late in the disease course, and such viruses may contribute to neurocognitive impairment. Here, we examine the CD4 binding properties of the viral Env protein to explore these two different entry phenotypes.
MeSH term(s)	Humans ; CD4 Antigens/metabolism ; CD4-Positive T-Lymphocytes ; Gene Products, env/metabolism ; HIV-1/physiology ; Macrophages/metabolism ; Receptors, CCR5/metabolism ; Viral Envelope Proteins/metabolism ; env Gene Products, Human Immunodeficiency Virus
Chemical Substances	CD4 Antigens ; Gene Products, env ; Receptors, CCR5 ; Viral Envelope Proteins ; gp160 protein, Human immunodeficiency virus 1 ; env Gene Products, Human Immunodeficiency Virus
Language	English
Publishing date	2024-03-01
Publishing country	United States
Document type	Journal Article
ZDB-ID	2557172-2
ISSN	2150-7511 ; 2161-2129
ISSN (online)	2150-7511
ISSN	2161-2129
DOI	10.1128/mbio.00321-24
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Article: A Comparative Study on the Pore Size Distribution of Different Indian Shale Gas Reservoirs for Gas Production and Potential CO2 Sequestration

Tripathy, Ashutosh / T. N. Singh / Vinoth Srinivasan

Energy & fuels. 2018 Feb. 21, v. 32, no. 3

2018

Abstract: A thorough knowledge on pore size distribution (PSD) is one of the fundamental requirements for characterization of shale gas reservoirs and for accurate estimation of their gas storage potential. However, being an important future source of energy need ... ...

Abstract	A thorough knowledge on pore size distribution (PSD) is one of the fundamental requirements for characterization of shale gas reservoirs and for accurate estimation of their gas storage potential. However, being an important future source of energy need in India, the pore size distribution of Indian shale gas systems is not scientifically well understood. In the present study, the nanoscale pore size distributions of prospective Indian shale basins, viz., Cambay, Cauvery, Krishna-Godavari (K-G), and Damodar valley (DV), were investigated using mercury injection porosimetry (MIP) and low-pressure gas adsorption (LP-N2 and LP-CO2) techniques. The study focused on identifying the priority basins for shale gas production which can be substituted for sequestration of CO2 based on their PSDs. The samples exhibited higher thermal maturity with increasing organic content. The chemical composition of the shale samples was inferred from XRD data, which depicted higher clay content. The prominent clay minerals identified muscovite, illite, and kaolinite, which are generally flaky in nature. These minerals contributed significantly to the pore size complexity of the studied shale samples. The experimental result suggested that the samples exhibited diversified pore size characteristics. The shales are chiefly bimodal, consisting of mesopores (2–50 nm) and micropores (<2 nm). The micropores were efficiently accessed using CO2, while N2 was effective on characterizing the mesopore region. MIP analysis was used to infer the pore throat area. The average pore diameter of samples ranged from 3.38 nm in Damodar valley to 3.94 nm in thr K-G basin, while Cauvery and Cambay basin samples possessed 3.88 and 3.86 nm, respectively. Both N2 and CO2 adsorption (type II and type I, respectively) suggested the presence of micropore infilling in all of the basin samples. However, Cauvery basin samples were inferred with higher mesopore content as compared to other basins and possessed higher quartz percentage, making it more appropriate for hydraulic fracturing. The Cauvery basin showed enrichment of larger pore sizes. The results signified that the Damodar valley shales have a higher affinity toward CO2 adsorption. This suggested that the basin could be a better host for future carbon storage, compared to other basin pore structures.
Keywords	adsorption ; basins ; carbon dioxide ; carbon sequestration ; chemical composition ; clay fraction ; energy ; fuels ; hydraulic fracturing ; illite ; kaolinite ; mercury ; micropores ; muscovite ; nitrogen ; porosimetry ; porosity ; quartz ; shale ; shale gas ; X-ray diffraction ; India
Language	English
Dates of publication	2018-0221
Size	p. 3322-3334.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	1483539-3
ISSN	1520-5029 ; 0887-0624
ISSN (online)	1520-5029
ISSN	0887-0624
DOI	10.1021/acs.energyfuels.7b04137
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Article ; Online: AlphaFold accurately predicts distinct conformations based on the oligomeric state of a de novo designed protein.

Cummins, Matthew C / Jacobs, Tim M / Teets, Frank D / DiMaio, Frank / Tripathy, Ashutosh / Kuhlman, Brian

Protein science : a publication of the Protein Society

2022 Volume 31, Issue 7, Page(s) e4368

Abstract: Using the molecular modeling program Rosetta, we designed a de novo protein, called SEWN0.1, which binds the heterotrimeric G protein ... ...

Abstract	Using the molecular modeling program Rosetta, we designed a de novo protein, called SEWN0.1, which binds the heterotrimeric G protein Gα
MeSH term(s)	Amino Acid Sequence ; Artificial Intelligence ; Models, Molecular ; Protein Conformation ; Proteins/chemistry
Chemical Substances	Proteins
Language	English
Publishing date	2022-06-27
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	1106283-6
ISSN	1469-896X ; 0961-8368
ISSN (online)	1469-896X
ISSN	0961-8368
DOI	10.1002/pro.4368
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Article: Probiotics: A Promising Candidate for Management of Colorectal Cancer.

Tripathy, Ashutosh / Dash, Jayalaxmi / Kancharla, Sudhakar / Kolli, Prachetha / Mahajan, Deviyani / Senapati, Shantibhusan / Jena, Manoj Kumar

Cancers

2021 Volume 13, Issue 13

Abstract: Colorectal cancer (CRC) is the World's third most frequently diagnosed cancer type. It accounted for about 9.4% mortality out of the total incidences of cancer in the year 2020. According to estimated facts by World Health Organization (WHO), by 2030, 27 ...

Abstract	Colorectal cancer (CRC) is the World's third most frequently diagnosed cancer type. It accounted for about 9.4% mortality out of the total incidences of cancer in the year 2020. According to estimated facts by World Health Organization (WHO), by 2030, 27 million new CRC cases, 17 million deaths, and around 75 million people living with the disease will appear. The facts and evidence that establish a link between the intestinal microflora and the occurrence of CRC are quite intuitive. Current shortcomings of chemo- and radiotherapies and the unavailability of appropriate treatment strategies for CRC are becoming the driving force to search for an alternative approach for the prevention, therapy, and management of CRC. Probiotics have been used for a long time due to their beneficial health effects, and now, it has become a popular candidate for the preventive and therapeutic treatment of CRC. The probiotics adopt different strategies such as the improvement of the intestinal barrier function, balancing of natural gut microflora, secretion of anticancer compounds, and degradation of carcinogenic compounds, which are useful in the prophylactic treatment of CRC. The pro-apoptotic ability of probiotics against cancerous cells makes them a potential therapeutic candidate against cancer diseases. Moreover, the immunomodulatory properties of probiotics have created interest among researchers to explore the therapeutic strategy by activating the immune system against cancerous cells. The present review discusses in detail different strategies and mechanisms of probiotics towards the prevention and treatment of CRC.
Language	English
Publishing date	2021-06-25
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2527080-1
ISSN	2072-6694
ISSN	2072-6694
DOI	10.3390/cancers13133178
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Article: Influence of convective hot air drying on physico-functional, thermo-pasting and antioxidant properties of elephant foot yam powder (

Srikanth, K S / Sharanagat, Vijay Singh / Kumar, Yogesh / Singh, Lochan / Suhag, Rajat / Thakur, Dhruv / Tripathy, Ashutosh

Journal of food science and technology

2021 Volume 60, Issue 3, Page(s) 879–888

Abstract: The present study focused on the effect of different drying temperatures (40, 50, 60 and 70 °C) and combination of pre-treatments: potassium metabisulphite (KMS), potassium metabisulphite + Citric acid + blanching (KCB)] on functional, thermo-pasting and ...

Abstract	The present study focused on the effect of different drying temperatures (40, 50, 60 and 70 °C) and combination of pre-treatments: potassium metabisulphite (KMS), potassium metabisulphite + Citric acid + blanching (KCB)] on functional, thermo-pasting and antioxidant properties of elephant foot yam (EFY) powder. Drying temperature and pretreatment reduces the water and oil absorption capacity, and the highest values were 2.34 g/g and 1.19 g/g for drying at 40 °C for the untreated sample, respectively. KMS pretreatment enhanced the bulk density, foaming capacity, emulsion capacity, and emulsion stability with an increase in drying temperature. Pasting temperature and viscosity decreased with an increase in drying temperature, and the maximum was observed at 40 °C for KMS pretreatment. Blanching increases the gelatinization temperature resulting in higher mid-and end-temperatures for KCB pretreatment. The antioxidant properties decreased with an increase in the drying temperature and were found to be minimal in the case of KCB treated samples.
Language	English
Publishing date	2021-02-28
Publishing country	India
Document type	Journal Article
ZDB-ID	242498-8
ISSN	0975-8402 ; 0022-1155
ISSN (online)	0975-8402
ISSN	0022-1155
DOI	10.1007/s13197-021-05015-6
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Article ; Online: A conserved set of mutations for stabilizing soluble envelope protein dimers from dengue and Zika viruses to advance the development of subunit vaccines.

Phan, Thanh T N / Hvasta, Matthew G / Kudlacek, Stephan T / Thiono, Devina J / Tripathy, Ashutosh / Nicely, Nathan I / de Silva, Aravinda M / Kuhlman, Brian

The Journal of biological chemistry

2022 Volume 298, Issue 7, Page(s) 102079

Abstract: Dengue viruses (DENV serotypes 1-4) and Zika virus (ZIKV) are related flaviviruses that continue to be a public health concern, infecting hundreds of millions of people annually. The traditional live-attenuated virus vaccine approach has been challenging ...

Abstract	Dengue viruses (DENV serotypes 1-4) and Zika virus (ZIKV) are related flaviviruses that continue to be a public health concern, infecting hundreds of millions of people annually. The traditional live-attenuated virus vaccine approach has been challenging for the four DENV serotypes because of the need to achieve balanced replication of four independent vaccine components. Subunit vaccines represent an alternative approach that may circumvent problems inherent with live-attenuated DENV vaccines. In mature virus particles, the envelope (E) protein forms a homodimer that covers the surface of the virus and is the major target of neutralizing antibodies. Many neutralizing antibodies bind to quaternary epitopes that span across both E proteins in the homodimer. For soluble E (sE) protein to be a viable subunit vaccine, the antigens should be easy to produce and retain quaternary epitopes recognized by neutralizing antibodies. However, WT sE proteins are primarily monomeric at conditions relevant for vaccination and exhibit low expression yields. Previously, we identified amino acid mutations that stabilize the sE homodimer from DENV2 and dramatically raise expression yields. Here, we tested whether these same mutations raise the stability of sE from other DENV serotypes and ZIKV. We show that the mutations raise thermostability for sE from all the viruses, increase production yields from 4-fold to 250-fold, stabilize the homodimer, and promote binding to dimer-specific neutralizing antibodies. Our findings suggest that these sE variants could be valuable resources in the efforts to develop effective subunit vaccines for DENV serotypes 1 to 4 and ZIKV.
MeSH term(s)	Antibodies, Neutralizing ; Antibodies, Viral ; Cross Reactions ; Dengue/prevention & control ; Dengue Virus/genetics ; Epitopes ; Humans ; Mutation ; Vaccines, Attenuated ; Vaccines, Subunit/genetics ; Viral Envelope Proteins/genetics ; Viral Vaccines/genetics ; Zika Virus/genetics ; Zika Virus Infection/prevention & control
Chemical Substances	Antibodies, Neutralizing ; Antibodies, Viral ; Epitopes ; Vaccines, Attenuated ; Vaccines, Subunit ; Viral Envelope Proteins ; Viral Vaccines
Language	English
Publishing date	2022-05-26
Publishing country	United States
Document type	Journal Article
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1016/j.jbc.2022.102079
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Article ; Online: Role of Natural Killer Cells during Pregnancy and Related Complications.

Mahajan, Deviyani / Sharma, Neeta Raj / Kancharla, Sudhakar / Kolli, Prachetha / Tripathy, Ashutosh / Sharma, Amarish Kumar / Singh, Sanjeev / Kumar, Sudarshan / Mohanty, Ashok Kumar / Jena, Manoj Kumar

Biomolecules

2022 Volume 12, Issue 1

Abstract: A high number of leucocytes reside in the human endometrium and are distributed differentially during the menstrual cycle and pregnancy. During early pregnancy, decidual natural killer (dNK) cells are the most common type of natural killer (NK) cells in ... ...

Abstract	A high number of leucocytes reside in the human endometrium and are distributed differentially during the menstrual cycle and pregnancy. During early pregnancy, decidual natural killer (dNK) cells are the most common type of natural killer (NK) cells in the uterus. The increase in the number of uterine NK (uNK) cells during the mid-secretory phase of the menstrual cycle, followed by further increase of dNK cells in early pregnancy, has heightened interest in their involvement during pregnancy. Extensive research has revealed various roles of dNK cells during pregnancy including the formation of new blood vessels, migration of trophoblasts, and immunological tolerance. The present review article is focused on the significance of NK cells during pregnancy and their role in pregnancy-related diseases. The article will provide an in-depth review of cellular and molecular interactions during pregnancy and related disorders, with NK cells playing a pivotal role. Moreover, this study will help researchers to understand the physiology of normal pregnancy and related complications with respect to NK cells, so that future research work can be designed to alleviate the complications.
MeSH term(s)	Decidua/immunology ; Female ; Humans ; Immune Tolerance ; Killer Cells, Natural/immunology ; Pregnancy ; Pregnancy Complications/immunology ; Trophoblasts/immunology
Language	English
Publishing date	2022-01-04
Publishing country	Switzerland
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2701262-1
ISSN	2218-273X ; 2218-273X
ISSN (online)	2218-273X
ISSN	2218-273X
DOI	10.3390/biom12010068
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