Archive 22/3/30

Monoclonal antibody reference standard with extensive glycan characterization: NISTmAb

Global Biologics, Science Division, United States Pharmacopeia, Rockville, MD 20852, USA has disclosed a monoclonal antibody reference standard, with extensive glycan characterization.

A monoclonal antibody reference standard, with extensive glycan characterization is available from the National Institute of Standards and Technology designated as NIST Reference Material 8671, NISTmAb.
The United States Pharmacopeia (USP) developed three monoclonal antibody reference standards (i.e., USP mAb 001 RS, USP mAb 002 RS, and USP mAb 003 RS) that may be used as control materials to demonstrate whether glycan characterization procedures provide an accurate result.
The USP mAb reference standards are different proteins of the same IgG type 1 subclass yet provide sufficient variability to examine a broad spectrum of glycan structures. Glycan characterization is commonly performed by analysis of glycans released from the protein backbone.

F(6)A2: core fucosylated biantennary,
F(6)A2B: core fucosylated biantennary with bisecting GlcNAc,
M5: five mannose on core GlcNAc,
F(6)A1G(4)1: core fucosylated monoantennary with β1-4 linked Galactose,
A2[6]G(4)1: biantennary with β1-4 linked Galactose attached to α1-6 linked core mannose,
F(6)A2[6]G(4)1: core fucosylated biantennary with β1-4 linked Galactose attached to α1-6 linked core mannose,
F(6)A2[3]G(4)1: core fucosylated biantennary with β1-4 linked Galactose attached to α1-3 linked core mannose,
F(6)A2[6]G(4)1Ga1: fucosylated bianntennary with a β1,4-linked galactose directly attached to the α1,6-linked core mannose, and an α1,3-linked galactose attached to the β1,4-linked galactose,
F(6)A2G(4)2: equal to G2F,

Targeting TGF-β1 pathway might be a promising approach to enhance immune checkpoint blockade PD-1/PD-L1 in nasopharyngeal carcinoma

A group from Department of Otolaryngology-Head and Neck Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China, etc. has reported that targeting TGF-β1 pathway might be a promising approach to enhance immune checkpoint blockade PD-1/PD-L1 in nasopharyngeal carcinoma (NPC).

It is well known that PD-L1, programmed death ligand-1, is a transmembrane glycoprotein expressed by tumor cells and PD-L1 inhibits T cells activity and promotes immune evasion through binding to its receptor PD-1 on T cells.

TGF-β is a multifunctional cytokine with an important role in both physiologic and pathologic processes, including cancer. The aberrantly upregulated production of TGF-β has been strongly implicated in tumor progression, angiogenesis, and metastasis, as well as immune escape. TGF-β has been regarded as a critical immunosuppressive cytokine, which suppresses the antitumor activity of effector cells, including CD8+ T cells, natural killer (NK) cells, and macrophages.

In this study, it was confirmed that PD-L1 was heavily glycosylated in NPC, and found that TGF-β1 played an important role in N-glycosylation of PD-L1.
Inhibition of TGF-β1 by a TGF-β type I receptor inhibitor (SB431542) reduced not only PD-L1 expression but also PD-L1 glycosylation. The reduction of PD-L1 glycosylation was confirmed by loss of PD-L1 binding ability to ConA lectin.

Authors found that the glycosylation of PD-L1 was affected by TGF-β1 through c-Jun/STT3A signaling pathway, suggesting that targeting TGF-β1 pathway might be a promising approach to enhance immune checkpoint blockade in NPC avoiding deglycosylatin of PD-L1.

Co-culture of NPC cells (5-8F cells) and Jurkat T cells, conducted with STT3A-knockdown (shSTT3Acells), SB431542 and tunicamycin (TM) pretreatments on T cells.

Alleviation of salinity stress in eggplants by using a biofertilizer (Bacillus subtilis, Pseudomonas sp., Trichoderma harzianum etc.)

A group from Department of Botany and Microbiology, Faculty of Science, Alexandria University, Alexandria 21511, Egypt, etc. has reported about alleviation of salinity stress in eggplants by using a biofertilizer.

Chemical fertilizers have been widely used to achieve maximum crop productivity in conventional agricultural systems. Nevertheless, when chemical fertilizers exceed the threshold level, they accelerate soil acidification, pollute groundwater, and harm the environment overall. It is recommended to apply biofertilizers as eco-friendly alternatives as they play a pivotal role in phosphate solubilization, nitrogen fixation, production of ammonia, enzymes, siderophores, and secretion of variable phytohormones. Furthermore, they exhibit biocontrol activity against a wide variety of phytopathogenic agents.

In this study, the effect of selected bioinoculum (plant growth promoting rhizobcteria and rhizo fungi) on salinity stress in eggplants (Solanum melongena L.) was investigated as a biofertilizer.

Comparative experimental conditions were as follows:
(C): one irrigated with distilled water as a control,
(I): the second one inoculated with the selected inoculum and irrigated with distilled water,
(S): the third one irrigated with 200 mM NaCl, and
(S + I): the fourth one irrigated with 200 mM NaCl and inoculated with selected inoculum.
Two strains of rhizobacteria (Bacillus subtilis and Pseudomonas sp.) and three strains of rhizofungi (Trichoderma harzianum, Aspergillus terrus, and Penicillium citrinum) were used as the inoculum (as a biofertilizer).

Salinity stress significantly decreased leaf area, root fresh and dry weights, in addition to root water content by (59.14, 95.87, 96.30, and 95.66%, respectively) compared to control, but the inoculating the soil of salt-stressed eggplants showed a significant increase in leaf area (LA), total fresh weight (TFW), total dry weight (TDW), and total water content(TWC) as shown below.

The gene expression of photosystem II D2, glutathione reductase, and glutathione S transferase was upregulated in inoculated salt-stressed eggplants with respect to stressed ones, that of protease I and protease II (cell-wall degrading enzymes to suppress pathogen growth) was also upregulated in inoculated salt-stressed eggplants with respect to stressed ones, and that of lipase was downregulated conversely in inoculated salt-stressed eggplants with respect to stressed ones.
These changes suggest that defense mechanism related genes are upregulated in inoculated eggplants.

Changes in gastric mucosal glycosylation before and after Helicobacter pylori Eradication: Studies using Lectin Microarray

A group from Department of Gastroenterology, Faculty of Medicine, Oita University, Oita, Japan, has reported about changes in gastric mucosal glycosylation before and after Helicobacter pylori Eradication.

It was found that the jacalin and MPA signals in the gastric antrum were significantly lower in the H. pylori infection group than in the gastric mucosa of the group without H. pylori infection, and that, 1 year after eradication, the signals returned to the levels seen without infection.
The LTL, SNA, SSA, and TJA-I signals in the gastric body were significantly increased in the H. pylori infection group and returned to the levels seen without infection 1 year after eradication.

MPA and jacalin are 85% homologous, and these lectins have particularly high specificity for the TF-antigen that is expressed in more than 85% of human carcinomas.
LTL binds to Lewisx, Lewisy and H-antigen type II, and has been reported to be a marker for cancer progression in bladder cancer cell lines.
SNA has been reported to be a marker for the diagnosis, metastasis, and prognosis of colorectal and pancreatic cancers and hepatocellular carcinoma. SNA has also been reported to be a diagnostic marker for pneumonia and a predictive marker for diabetic nephropathy progression. SNA, SSA, and TJA-I have the same glycan binding specificity, α2-6Sia.

The results of this study showed that the signal levels for lectin binding to glycans resulting from H. pylori infection returned to the levels seen without infection as a result of H. pylori eradication. Thus, the data are intriguing in that they indicate that glycosylation is reversible.

How Bacillus subtilis manage to efficiently migrate in the semisolid or solid soil environment?

A group from Institute of Plant Protection, Tianjin Academy of Agricultural Sciences, Tianjin, China, etc. has reported on a novel sucrose induced signaling cascade promoting Bacillus subtilis rhizosphere colonization.

It has been known that Bacillus subtilis is an excellent biological control agent, capable of suppressing a number of soil-borne phytopathogens. But, how do such beneficial bacteria recognize the signals from the plant host, colonize the roots, and ultimately establish an intimate relationship with the plant? One of the specific focuses is on plant root-released nutrients and their impact on the rhizobacteria. Among those root-released nutrients, sucrose is found most abundantly released into the rhizosphere. Sucrose is uniquely important to the plants since photosynthetic plants primarily use sucrose as a fixed carbon transport and storage mechanism among different tissues.

It is clearly shown in the figure below that sucrose induces more robust root colonization on tomato roots by Bacillus subtilis in the presence of sucrose than several other root-secreted sugars.

A red fluorescence-labeled Batillus subtilis strain was used with different sugars (Fru: fructose, Glc: glucose, Mal: maltose, and Suc: sucrose)

Further, in the rhizosphere soil with sucrose addition, the relative abundance of Bacillus reached ~10.1%, while this ratio was only ~0.1% without sucrose addition, indicating a 100-fold increase in the abundance of the native Bacillus species in the rhizosphere stimulated by sucrose. In addition to the strong positive influence of sucrose on the prevalence of Bacillus, the relative low abundance of Pseudomonas (0.05%) was also elevated to 2.7% when inoculated with the wild type B. subtilis cells and further increased to 12.5% with simultaneous B. subtilis inoculation and sucrose supplementation. It was also shown that supplementation of sucrose formula improved the suppression efficiency against the soil-borne disease caused by Fusarium oxysporum sp. Lycopersici (Fusarium wilt) .

Big question is how B. subtilis manage to efficiently migrate in the semisolid or solid soil environment?

Regarding this question, authors has shown that sucrose activates a signaling cascade to trigger solid surface motility leading to effective root colonization by B. subtilis. That is, sucrose initiates biosynthesis of levan, and levan is further hydrolyzed into levanoligosaccharides, and finally induces strong production of surfactin from B. subtilis resulting in acceleration of solid surface motility.

Activation of the lectin pathway plays a vital role in the development of Idiopathic Membranous Nephropathy

Ther are two types of Membranous nephropathy (MN), idiopathic MN (iMN) and atypical MN (aMN). A group from Department of Nephrology, Peking University People’s Hospital, Beijing, China has reported that activation of the mannose-binding lectin (MBL) pathway plays a vital role in the development of iMN.

There are three major complement pathways include the classical pathway, mannose-binding lectin (MBL) pathway, and alternative pathway. C4d is generated in both the classical and mannose-binding lectin complement pathways. C1q is the major precursor of classical complement activation.
The presence of both C4d and C1q indicates the activation of the classical pathway, but
identification of C4d without C1q is more consistent with MBL pathyway, and
C4d and C1q are both absent in alternative complement activation.

The renal pathology of iMN is characterized by the deposition of predominant IgG4 with low amounts of IgG1 and IgG3. IgG4 does not activate the classical complement pathway. C4d is detectable in essentially 100% of patients with iMN which was absent in alternative complement activation. These observations suggest that the MBL-initiated complement pathway may be the predominant complement activation in iMN. On the other hand, aMN characterized by cells proliferation, multi-site immune complex deposition, ‘full house’ in immunofluorescence, including IgA, IgG, IgM, C3, C1q positive, implies that the pathophysiological process involves complex complement system activation.

Lactobacillus crispatus Strain KT-11 S-Layer Protein can inhibit Rotavirus Infection: Sialic acid would be deeply involved

A group from Shinshu University, Nagano, Japan, etc. has reported that Lactobacillus crispatus Strain KT-11 S-Layer Protein can inhibit Rotavirus Infection and sialic acid could be involved in initiation of virus infection.

Lactic acid bacteria, including the genus Lactobacillus play a crucial role in the production of fermented dairy products such as cheese, yoghurt, and fermented milk. In recent years, there has been considerable focus on the action of the S-layer protein (SLP) of lactic acid bacteria as an antiviral component.

The effect of Lactobacillus crispatus KT-11 SLP on the infection of Rotavirus DS-1 strain in Caco-2 cells is shown below. DS-1 infection was significantly suppressed by pre-infection treatment with KT-11 SLP in a concentration-dependent manner. Conversely, KT-11 SLP did not suppress the infection of the Rotavirus Wa strain even after pre-infection treatment at 100 μg/mL.

The entry of rotavirus into cells is a complex multistep process, in which different domains of rotavirus surface proteins interact with cell-surface molecules that function as receptors for adhesion and entry. Among them, several carbohydrates, such as terminal sialic acids and histo-blood group antigens, have been reported to be involved in rotavirus attachment to target cells. As shown above, KT-11 SLP significantly inhibited the infection of the DS-1 strain in Caco-2 cells in a dose-dependent manner. The initial interactions of human rotavirus strains with host cells is dependent on the VP4 genotype. According to the classification based on the molecular properties of VP4 (P-types), the DS-1 strain is classified as the P[4] genotype. Increasing evidence indicates that the P[4] genotype rotaviruses, including the DS-1 strain, use H-type 1 and Lewis-b antigens for infection. Actually, H-type 1 and Lewis-b antigens have been reported in Caco-2 cells. This result suggests that the possibility of that DS-1 infection is inhibited by competitive binding of KT-11 SLP to these antigens. However, contrary to the expectations, infection of the Wa strain, another dominant P[8] subtype reported using the same H-type antigen and Lewis-b for infection, was not inhibited in the presence of KT-11 SLP.

Recently, it was reported that DS-1 strain infection was significantly inhibited by 3′-sialyl lactose and 6′-sialyl lactose, whereas the Wa strain was inhibited by 2′-fucosyl lactose. Therefore, compounds containing sialic acid could be deeply involved in the infection of DS-1 strain.

Changes in belowground Fungal flora in wheat field under long-term fertilization

A group from State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China, etc. has reported about changes in belowground fungal flora in wheat field under long-term fertilization.
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In this paper, new findings are reported from a long-running fertilization experiment in wheat field (more than 35 years) covering the following conditions: NPK fertilizer, NPK and cow manure (NPK+CM), NPK and pig manure (NPK+PM), NPK and wheat straw (NPK+WS), and no fertilizer (Control).

At the phylum level, Ascomycota, Basidiomycota, and Mortierellomycota dominated the fungal community, irrespective of the treatments and habitats, together accounting for over 80% of the total sequences obtained. Compared with the other treatments, NPK+PM treatment had the highest relative abundance of Ascomycota (96.9%, 87.1%, and 91.1% in root endosphere, rhizosphere soil, and bulk soil, respectively)

Then, the correlation between alpha diversity and physiochemical variables (such as Total P, Total C, Total N, Total K, P, Ca, Mg, Na, Fe, Mn etc.) was analyzed. The random forest analysis revealed that phosphorus and Zn always were the best predictors of diversity changes in root endosphere, rhizosphere soil, and bulk soil. There were significant correlations between phosphorus and alpha diversity in root endosphere, and between Zn and alpha diversity in rhizosphere soil and bulk soil. The higher the P and Zn concentration, the lower the alpha diversity in rhizosphere fungi.

This can be considered as follows. Pig manure had higher phosphorus content than cow pig manure and after adding pig manure, the phosphorus content of soils and wheat roots was significantly higher than that of cow manure addition. Hence, long-term fertilization, especially pig manure application, can supply adequate and readily accessible phosphorus and phosphorus-like nutrients across soil to roots, which may lessen the dependence of crops on microbiota, thereby lessening total fungal diversity probably. Similar to phosphorus, it is reasonable to link the competition for resources and survival among fungal communities to Zn-induced changes in fungal diversity.

Antagonist fungi isolated from soils of the rhizosphere of tomato crops against Sclerotium rolfsii: the causative agent of white rot

A group from Laboratoire Biologie et Santé, UFR Biosciences, Université Félix Houphouët-Boigny d’Abidjan (UFHB), Côte d’Ivoire, etc. has reported about antagonist fungi isolated from soils of the rhizosphere of tomato crops against Sclerotium rolfsii.

S. rolfsii is the causative agent of white rot is one of the destructive pathogens of nightshade crops. In Côte d’Ivoire, this fungal pathogen constitutes a major constraint for the cultivation of tomato (Solanum lycopersicum) with 41.01% crop losses in humid forest areas.

Antifungal activity of soil fungi against S. rolfsii
The screening carried out from the 153 soil-based fungal isolates by means of a direct confrontation test on the PDA medium made it possible to select 10 fungi which inhibit the growth of S. rolfsii. The percentages of inhibition ranged from 27.06 to 60.59%. The mechanisms of action observed during this inhibition were competition and antibiosis. The former is caused by the rapid growth of the antagonist will slow down or inhibit the growth of the pathogen, and the latter is caused by inhibiting biological activity of the pathogen. Molecular identification (ITS) of these antagonist fungi revealed that the isolates belonged to the genera Talaromyces sp. (n = 4), Trichoderma sp. (n = 3), Penicillium sp. (n = 2) and Clonostachys sp. (n = 1).

Among 10 antagonost fung, Talaromyces purpureogenus and Talaromyces assiutensis exerted the antibiosis mechanism.
T. purpureogenus secreted Mitorubrin and mitorubinol, and
T. assiutensis secreted Spiculisporic acid as molecules responsible for inhibiting S. rolfsii.

An ACE2-Fc fusion protein could be a good therapeutic drug overcoming viral escape of SARS-CoV-2 variants

A group from SystImmune Inc., 15318 NE 95th St., Redmond, WA, 98052, USA, etc. has reported that ACE2-Fc fusion protein could be a good therapeutic drug overcoming viral escape of SARS-CoV-2 variants.

Interestingly, the pseudovirus neutralization data using the ACE2-Fc fusion protein named SI-F09 (shown below) shows that the higher the binding strength between SARS-CoV-2 RBD and ACE2, the lower the IC50 values. In other words, the IC50 value for WT is the highest among SARS-CoV-2 variants imcruding Omicron.

In the meantime, a phase I clinical trial evaluating safety of SI–F019 in healthy patients has been conducted ( Identifier: NCT04851444) and is being analyzed in preparation for future clinical studies.

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