Simultaneous Cell Disruption and Aqueous Two-Phase Extraction for Isolation of Intracellular Recombinant Proteins

A new technique was developed for the integrated processing of cell disruption and aqueous two-phase extraction in a high-speed bead mill to separate intracellular proteins from microbial cells. The process was named as simultaneous cell disruption and aqueous two-phase extraction (SDATE). Advantages, such as high cell disruption efficiency, biochemical activities preservation of proteins, cell debris elimination, and prelimiary purification of the target protein were being claimed. When this technique was employed for isolating recombinant Tumor Necrosis Factor (TNF) from E. coli, overall protein concentration and TNF activity were found to have been increased. More than 95% of TNF was partitioned into the top phase and all cell debris were in the bottom phase. The partition coefficient was greater than 3 and the TNF purification factor was greater than 6. It is shown that less separation steps were being utilized in the new technique, meaning a reduction in separation time and less process extractors required.

Engineered extracellular vesicles enable high-efficient delivery of intracellular therapeutic proteins

Developing an intracellular delivery system is of key importance in the expansion of protein-based therapeutics acting on cytosolic or nuclear targets.Recently,extracellular vesicles(EVs)have been exploited as next-generation delivery modalities due to their natural role in intercellular communication and biocompatibility.However,fusion of protein of interest to a scaffold represents a widely used strategy for cargo enrichment in EVs,which could compromise the stability and functionality of cargo.Herein,we report intracellular delivery via Ev-based approach(IDEA)that efficiently packages and delivers native proteins both in vitro and in vivo without the use of a scaffold.As a proof-of-concept,we applied the IDEA to deliver cyclic GMP-AMP synthase(cGAS),an innate immune sensor.The results showed that cGAS-carrying EVs activated interferon signaling and elicited enhanced antitumor immunity in multiple syngeneic tumor models.Combining cGAS EVs with immune checkpoint inhibition further synergistically boosted antitumor efficacy in vivo.Mechanistically,scRNA-seq demonstrated that cGAS EVs mediated significant remodeling of intratumoral microenvironment,revealing a pivotal role of infiltrating neutrophils in the antitumor immune milieu.Collectively,IDEA,as a universal and facile strategy,can be applied to expand and advance the developmentof protein-based therapeutics.

Effect of Low Dose Radiation on Intracellular Calcium and Protein Kinase C in Lymphocytes

It is first reported in the present paper that whole-body irradiation (WBI) with low dose X-rays could increase intracellular calcium ions ([Ca2+]i) and stimulate protein kinase C (PKC) activity of mouse lymphocytes. Following WBI of male Kunming micc With 75 mGy X-rays at a dose rate of 12.5 mGy/min the mobilization of [Ca2+]i with Con A in CD4+ and CD8+ Cells in the thymus and spleen was potentiated and the amplitude of [Ca2+], mobilization in thymocytes in response to anti-CD3 monoclonal antibody increased with time from 4 to 24 h following low dose radiation. The PKC activity in the homogenate of spleen was markedly stimulated 12 h after WBl with 75 mGy, reaching its peak value at 24-48 h and coming down to lower than normal on day 7. However, the PKC activity in the separated T lymphocytes reached its peak value at 12 h and that in the B lymphocytes reached its peak value on day 4, both coming down to below control on day 7. The implications of this facilitation of signal transduction in T lymphocytes in the mechanism of immunoenhancement after low dose radiation were discussed

Two memory associated genes regulated by amyloid precursor protein intracellular domain Novel insights into the pathogenesis of learning and memory impairment in Alzheimer's disease

In this study, we employed chromatin immunoprecipitation, a useful method for studying the locations of transcription factors bound to specific DNA regions in specific cells, to investigate amyloid precursor protein intracellular domain binding sites in chromatin DNA from hippocampal neurons of rats, and to screen out five putative genes associated with the learning and memory functions. The promoter regions of the calcium/calmodulin-dependent protein kinase II alpha and glutamate receptor-2 genes were amplified by PCR from DNA products immunoprecipitated by amyloid precursor protein intracellular domain. An electrophoretic mobility shift assay and western blot analysis suggested that the promoter regions of these two genes associated with learning and memory were bound by amyloid precursor protein intracellular domain （in complex form）. Our experimental findings indicate that the amyloid precursor protein intracellular domain is involved in the transcriptional regulation of learning- and memory-associated genes in hippocampal neurons. These data may provide new insights into the molecular mechanism underlying the symptoms of progressive memory loss in Alzheimer＇s disease.

Intracellular sorting pathways of the amyloid precursor protein provide novel neuroprotective strategies

Alzheimer＇s disease（AD）is the most common cause of senile dementia.It is characterized by the formation of plaques mainly composed of the amyloid-beta peptide（Aβ）.Diverse lines of evidence support the notion that accumulation of Aβis a primary cause of AD pathogenesis（Huang and Mucke,2012）.Amyloid precusor protein（APP）processing is dependent on its subcelluar trafficking pathway：Aβis derived from APP by proteolyric processing.

COVID-19,liver dysfunction and pathophysiology:A conceptual discussion

The intra and extracellular pathways of hepatic injury by coronavirus disease 2019(COVID-19)are still being studied.Understanding them is important to treat this viral disease and other liver and biliary tract disorders.Thus,this paper aims to present three hypotheses about liver injury caused by COVID-19:(1)The interactions between severe acute respiratory syndrome coronavirus 2 spike protein and membrane receptors in the hepatocyte;(2)The dysbiosis and"gutliver axis"disruption in patients with serious clinical presentations of COVID-19;and(3)The inflammatory response exacerbated through the production of interleukins such as interleukin-6.However,despite these new perspectives,the pathophysiological process of liver injury caused by COVID-19 is still complex and multifactorial.Thus,understanding all these variables is a challenge to science but also the key to propose individualized and effective patient therapies.

Wireless Electrochemical Visualization of Intracellular Antigens in Single Cells

Electrochemical microscopy has been developed in the past decades for imaging biomolecules in single cells;however,electrochemical visualization of an intracellular protein in one cell is difficult.Herein,for the first time,we have developed a model whereby the protein,KDM1/LSD1 antigen,in the nucleus of a single MCF 7 cell could be visualized using wireless bipolar electrochemiluminescence(BPEECL).Submicron-sized,single-walled carbon nanotubes were linked with anti-KDM1/LSD1 antibodies and loaded into the cells for the recognition of the corresponding KDM1/LSD1 antigen.With a low electric field of 1000 V/cm,L012,a luminol analog,was oxidized electrochemically at one end of the nanotube that emitted light for the wireless visualization of its location.The significant drop in the applied voltage permitted the observation of apparent ECL emission inside the nucleus of a single cell,supporting the electrochemical imaging of intracellular KDM1/LSD1 antigen at a single-cell level.This work solves a long-standing task in the field of electroanalysis for intracellular wireless visualization,which should advance the development of electrochemistry in single-cell analysis.

Targeted and intracellular delivery of protein therapeutics by a boronated polymer for the treatment of bone tumors

The treatment of malignant bone tumors by chemotherapeutics often receives poor therapeutic response due to the specific physiological bone environment,and thus calls for the development of new therapeutic options.Here,we reported a bone-targeted protein nanomedicine for this purpose.Saporin,a toxin protein,was co-assembled with a boronated polymer for intracellular protein delivery,and the formed nanoparticles were further coated with an anionic polymer poly(aspartic acid)to shield the positive charges on nanoparticles and provide the bone targeting function.The prepared ternary complex nanoparticles showed high bone accumulation both in vitro and in vivo,and could reverse the surface charge property from negative to positive after locating at tumor site triggered by tumor extracellular acidity.The boronated polymer in the de-shielded nanoparticles further promote intracellular delivery of saporin into tumor cells,exerting the anticancer activity of saporin by inactivation of ribosomes.As a result,the bone-targeted and saporin-loaded nanomedicine could kill cancer cells at a low saporin dose,and efficiently prevented the progression of osteosarcoma xenograft tumors and bone metastatic breast cancer in vivo.This study provides a facile and promising strategy to develop protein-based nanomedicines for the treatment of malignant bone tumors.

Detection of lntraceUular Proteins and Biomarkers Using Modified Silica Nanoparticles and Flow Cytometry In vitro

In this paper, antibody-modified silica nanoparticles were successfully synthesized, and their average di- ameter was （1094-9） nm. These particles were mixed with cell extracts to target a protein, then, an antibody labeled with fluorescein isothiocyanate（FITC） was added to form FITC-labeled nanoparticle complexes and the product was analyzed using flow cytometry. The results confirm that the intracellular proteins and biomarkers were precisely and sensitivity detected by the novel method in vitro. The FITC intensity of Akt antibody-conjugated particles was in- creased ≥ 10-fold compared with that of control samples in MCF-7 cells. Furthermore, it can also simultaneously measure several proteins when modified with different antibodies.

Novel Adaptors of Amyloid Precursor Protein Intracellular Domain and Their Functional Implications

Amyloid precursor protein intracellular domain （AICD） is one of the potential candidates in deciphering the complexity of Alzheimer＇s disease. It plays important roles in determining cell fate and neurodegeneration through its interactions with several adaptors. The pres- ence or absence of phosphorylation at specific sites determines the choice of partners. In this study, we identified 20 novel AICD- interacting proteins by in vitro pull down experiments followed by 2D gel electrophoresis and MALDI-MS analysis. The identified proteins can be grouped into different functional classes including molecular chaperones, structural proteins, signaling and transport molecules, adaptors, motor proteins and apoptosis determinants. Interactions of nine proteins were further validated either by colocal- ization using confocal imaging or by co-immunoprecipitation followed by immunoblotting. The cellular functions of most of the proteins can be correlated with AD. Hence, illustration of their interactions with AICD may shed some light on the disease pathophysiology.

Relationship between Two Blood Stasis Syndromes and Inflammatory Factors in Patients with Acute Coronary Syndrome

Objective： To investigate the relationship between inflammatory factors and two Chinese medicine（CM） syndrome types of qi stagnation and blood stasis（QSBS） and qi deficiency and blood stasis（QDBS） in patients with acute coronary syndrome（ACS）. Methods： Sixty subjects with ACS, whose pathogenesis changes belongs to qi disturbance blood stasis syndrome, were divided into 2 groups： 30 in the QSBS group and 30 in the QDBS group. The comparative analysis on them was carried out through comparing general information, coronary angiography and inflammatory factors including intracellular adhesion molecule-1（ICAM-1）, chitinase-3-like protein 1（YKL-40） and lipoprotein-associated phospholipase A2（Lp-PLA2）. Results： Compared with the QSBS group, Lp-PLA2 and YKL-40 levels in the QDBS group showed no-significant difference（P〉0.05）; ICAM-1 was significantly higher in the QDBS group than in the QSBS group in the pathological processes of qi disturbance and blood stasis syndrome of ACS（P〈0.05）. Conclusion： Inflammatory factor ICAM-1 may be an objective basis for syndrome typing of QSBS and QDBS, which provides a research direction for standardization research of CM syndrome types.