Engineering multi-functional protein biologics through PAL-mediated hydrazide ligation

Attributed to the -effects, hydrazides are nucleophilic despite being non-basic. Herein, we report that the hydrazide nucleophiles are effective acyl acceptors in the ligation reactions catalyzed by peptidyl asparaginyl ligases (PALs). Because hydrazides are easily functionalizable, hydrazide ligation is highly versatile. Interestingly, the linkages formed with the hydrazide substrates have varying degrees of liability toward the PAL enzyme, with some being remarkably resistant and thus ensuring nearly irreversible ligation. Using the hydrazide ligation method, we labelled an EGFR-targeting affibody-Fc fusion protein with various functional moieties to generate selective and potent cancer-imaging and therapeutic agents. Irreversible hydrazide ligation also allowed a sequential ligation scheme to be conducted on a protein. Using this scheme, quadruple FITC labels were introduced onto the N- and C-termini of the affibody-Fc protein to yield a bi-specific engager for Car-NK cell therapy. Our work expands the substrate scope of PAL enzymes and further point to their promise as a precision manufacturing tool for multi-functional protein biologics.

Identification of The Intersegmental Plane By Arterial-Ligation Method During Thoracoscopic Segmentectomy

Purpose The purpose of this study is to explore the feasibility of identifying the intersegmental plane by arterial ligation alone during thoracoscopic anatomical segmentectomy. Methods We selected 35 patients with peripheral small lung nodules who underwent thoracoscopic anatomical segmentectomy between May and December 2020. First, the targeted segmental arteries were distinguished and ligated during the operation. Then, bilateral pulmonary ventilation was performed with pure oxygen to fully inflate the entirety pulmonary lobes. After waiting for a while, the intersegmental plane appeared. Finally, the intersegmental plane was observed using thoracoscopy after indocyanine green was injected into the peripheral vein. The intersegmental planes determined by these two methods were compared. Results Thirty-four patients underwent segmental resection and one patient finally underwent lobectomy. The intersegmental planes were successfully observed in all patients using the arterial ligation method. The time from contralateral pulmonary ventilation to the appearance of the intersegmental plane was 13.7±3.2 min (6-19 min). The intersegmental planes determined by the arterial ligation method and the fluorescence method were comparable. After the operation, CT examinations showed that the remaining lung segments of all patients were well inflated. The mean duration of closed thoracic drainage was 3.1±0.9 days. Conclusion The arterial ligation method can be used to determine the intersegmental plane in anatomical segmentectomy. The method is feasible, reliable, and safe.

A protocol for the reduction of applied torque on parent vessel duringelastase-induced aneurysm formation using rabbit animal models

Endovascular therapies for intracranial aneurysms requires animal models for testing the safety andeffectiveness prior to translation to the clinic. Rabbits combined with the elastase and right commoncarotid artery (RCCA) ligation methods is currently a widely used animal model for endovascular de-vice testing. However, the injection of elastase utilizing angiocatheters may potentially exerts adversetorque to the parent vessel and the optimal aneurysm creation period has not been well investigated.In this study, we present a modification to the elastase/RCCA-ligation method by replacing the angio-catheter with a butterfly catheter. Formation of saccular aneurysms was introduced in New Zealandwhite rabbits (n=6), and were maintained for 2, 4 and 6 weeks. The formed aneurysms exhibitedan elongated geometry and were stable during the study period. We found that the modification inthe animal surgery procedure provides improved manipulation of the surgical area, prolonged injec-tion of elastase, and effective degradation of the vascular elastic lamina. Compared to the traditionalelastase/RCCA-ligation method, the present technique can more effectively reduce unwanted injury tothe parent vessel and, therefore, improved stability of the vasculature for testing the efficacy of newlydeveloped endovascular embolization devices.

An APEX2 proximity ligation method for mapping interactions with the nuclear lamina

The nuclear lamina (NL) is a meshwork found beneath the inner nuclear membrane. The study of the NL is hindered by the insolubility of the meshwork and has driven the development of proximity ligation methods to identify the NL-associated/proximal proteins, RNA, and DNA. To simplify and improve temporal labeling, we fused APEX2 to the NL protein lamin-B1 to map proteins, RNA, and DNA. The identified NL-interacting/proximal RNAs show a long 3′ UTR bias, a finding consistent with an observed bias toward longer 3′ UTRs in genes deregulated in lamin-null cells. A C-rich motif was identified in these 3′ UTR. Our APEX2-based proteomics identifies a C-rich motif binding regulatory protein that exhibits altered localization in lamin-null cells. Finally, we use APEX2 to map lamina-associated domains (LADs) during the cell cycle and uncover short, H3K27me3-rich variable LADs. Thus, the APEX2-based tools presented here permit identification of proteomes, transcriptomes, and genome elements associated with or proximal to the NL.

Design, Synthesis and Characterization of Cyclic NU172 Analogues: A Biophysical and Biological Insight

NU172—a 26-mer oligonucleotide able to bind exosite I of human thrombin and inhibit its activity—was the first aptamer to reach Phase II clinical studies as an anticoagulant in heart disease treatments. With the aim of favoring its functional duplex-quadruplex conformation and thus improving its enzymatic stability, as well as its thrombin inhibitory activity, herein a focused set of cyclic NU172 analogues—obtained by connecting its 5′- and 3′-extremities with flexible linkers—was synthesized. Two different chemical approaches were exploited in the cyclization procedure, one based on the oxime ligation method and the other on Cu(I)-assisted azide-alkyne cycloaddition (CuAAC), affording NU172 analogues including circularizing linkers with different length and chemical nature. The resulting cyclic NU172 derivatives were characterized using several biophysical techniques (ultraviolet (UV) and circular dichroism (CD) spectroscopies, gel electrophoresis) and then investigated for their serum resistance and anticoagulant activity in vitro. All the cyclic NU172 analogues showed higher thermal stability and nuclease resistance compared to unmodified NU172. These favorable properties were, however, associated with reduced—even though still significant—anticoagulant activity, suggesting that the conformational constraints introduced upon cyclization were somehow detrimental for protein recognition. These results provide useful information for the design of improved analogues of NU172 and related duplex-quadruplex structures.

Gastroprotective effect of leaves of Breyniavitis-Idaea

Due to the lure backs of synthetic drugs that are being used medical field has turned over to the traditional medicine which are devoid of side effects and major adverse effects. So that Herbal drugs were seen as probable replacements for the handling of PUD without showing side effects and equaling the treatment efficacy. The literature review on investigations of antiulcer activity of various plant drug, the present investigation was carried out to investigate the antiulcer potential, chemical constituents present in the methanol extract of traditional plant Breyniavitis-idaea. Gastric ulcers in experimental animals were brought by four different models like Ethanol(Alcohol) induced, NSIDS (Indomethacin) induced, Pylorus ligation method and Cold resistant stress induced method by comparing with the standard drug namely omeprazole (20mg/kg) which exhibited the dose dependent capacity of the extract (125mg/kg,250mg/kg, 500mg/kg) and also the biochemical parameters like ALP, GSH, pH and Gastric volume contents were estimated in all the selected groups (Design of Experiment). The results obtained from the study has helped to identify that 500mg/kg of plant extract has gastro protective effect in all the chosen models in comparison to the omeprazole (20mg/kg) standard drug. Owing to the prevalence of different phytoconstituents like poly phenols and flavonoids shown the dose dependent potent gastroprotective activity.