An Innovative Mitochondrial-targeted Gene Therapy for Cancer Treatment.

Targeting cancer cell mitochondria holds great therapeutic promise, yet current strategies to specifically and effectively destroy cancer mitochondria in vivo are limited. Here, we introduce mLumiOpto, an innovative mitochondrial-targeted luminoptogenetics gene therapy designed to directly disrupt the inner mitochondrial membrane (IMM) potential and induce cancer cell death. We synthesize a blue light-gated channelrhodopsin (CoChR) in the IMM and co-express a blue bioluminescence-emitting Nanoluciferase (NLuc) in the cytosol of the same cells. The mLumiOpto genes are selectively delivered to cancer cells in vivo by using adeno-associated virus (AAV) carrying a cancer-specific promoter or cancer-targeted monoclonal antibody-tagged exosome-associated AAV. Induction with NLuc luciferin elicits robust endogenous bioluminescence, which activates mitochondrial CoChR, triggering cancer cell IMM permeability disruption, mitochondrial damage, and subsequent cell death. Importantly, mLumiOpto demonstrates remarkable efficacy in reducing tumor burden and killing tumor cells in glioblastoma or triple-negative breast cancer xenografted mouse models. These findings establish mLumiOpto as a novel and promising therapeutic strategy by targeting cancer cell mitochondria in vivo.

Advanced biomanufacturing and evaluation of adeno-associated virus.

Recombinant adeno-associated virus (rAAV) has been developed as a safe and effective gene delivery vehicle to treat rare genetic diseases. This study aimed to establish a novel biomanufacturing process to achieve high production and purification of various AAV serotypes (AAV2, 5, DJ, DJ8). First, a robust suspensive production process was developed and optimized using Gibco Viral Production Cell 2.0 in 30-60 mL shaker flask cultures by evaluating host cells, cell density at the time of transfection and plasmid amount, adapted to 60-100 mL spinner flask production, and scaled up to 1.2-2.0-L stirred-tank bioreactor production at 37 °C, pH 7.0, 210 rpm and DO 40%. The optimal process generated AAV titer of 7.52-8.14 × 1010 vg/mL. Second, a new AAV purification using liquid chromatography was developed and optimized to reach recovery rate of 85-95% of all four serotypes. Post-purification desalting and concentration procedures were also investigated. Then the generated AAVs were evaluated in vitro using Western blotting, transmission electron microscope, confocal microscope and bioluminescence detection. Finally, the in vivo infection and functional gene expression of AAV were confirmed in tumor xenografted mouse model. In conclusion, this study reported a robust, scalable, and universal biomanufacturing platform of AAV production, clarification and purification.

Characterization of the far-red fluorescent probe MitoView 633 for dynamic mitochondrial membrane potential measurement.

Introduction: MitoView 633, a far-red fluorescent dye, exhibits the ability to accumulate within mitochondria in a membrane potential-dependent manner, as described by the Nernst equation. This characteristic renders it a promising candidate for bioenergetics studies, particularly as a robust indicator of mitochondrial membrane potential (DYm). Despite its great potential, its utility in live cell imaging has not been well characterized. Methods: This study seeks to characterize the spectral properties of MitoView 633 in live cells and evaluate its mitochondrial staining, resistance to photobleaching, and dynamics during DYm depolarization. The co-staining and imaging of MitoView 633 with other fluorophores such as MitoSOX Red and Fluo-4 AM were also examined in cardiomyocytes using confocal microscopy. Results and Discussion: Spectrum analysis showed that MitoView 633 emission could be detected at 660 ± 50 nm, and exhibited superior thermal stability compared to tetramethylrhodamine methyl ester (TMRM), a commonly used DYm indicator, which emits at 605 ± 25 nm. Confocal imaging unequivocally illustrated MitoView 633's specific localization within the mitochondrial matrix, corroborated by its colocalization with MitoTracker Green, a well-established mitochondrial marker. Furthermore, our investigation revealed that MitoView 633 exhibited minimal photobleaching at the recommended in vitro concentrations. Additionally, the dynamics of MitoView 633 fluoresce during carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, a mitochondrial uncoupler)-induced DYm depolarization mirrored that of TMRM. Importantly, MitoView 633 demonstrated compatibility with co-staining alongside MitoSOX Red and Fluo-4 AM, enabling concurrent monitoring of DYm, mitochondrial ROS, and cytosolic Ca2+ in intact cells. Conclusion: These findings collectively underscore MitoView 633 as a superb molecular probe for the singular or combined assessment of DYm and other indicators in live cell imaging applications.

A CD276-Targeted Antibody-Drug Conjugate to Treat Non-Small Lung Cancer (NSCLC).

Non-small cell lung cancer (NSCLC) patients, accounting for approximately 85% of lung cancer cases, are usually diagnosed in advanced stages. Traditional surgical resection and radiotherapy have very limited clinical benefits. The objective of this study was to develop and evaluate a targeted therapy, antibody-drug conjugate (ADC), for NSCLC treatment. Specifically, the CD276 receptor was evaluated and confirmed as an ideal surface target of NSCLC in the immunohistochemistry (IHC) staining of seventy-three patient tumor microarrays and western blotting analysis of eight cell lines. Our anti-CD276 monoclonal antibody (mAb) with cross-activity to both human and mouse receptors showed high surface binding, effective drug delivery and tumor-specific targeting in flow cytometry, confocal microscopy, and in vivo imaging system analysis. The ADC constructed with our CD276 mAb and payload monomethyl auristatin F (MMAF) showed high anti-NSCLC cytotoxicity to multiple lines and effective anti-tumor efficacy in both immunocompromised and immunocompetent NSCLC xenograft mouse models. The brief mechanism study revealed the integration of cell proliferation inhibition and immune cell reactivation in tumor microenvironments. The toxicity study did not detect off-target immune toxicity or peripheral toxicity. Altogether, this study suggested that anti-CD276 ADC could be a promising candidate for NSCLC treatment.

Optogenetic Studies of Mitochondria.

While optogenetic approaches have been widely used for remote control of cell membrane excitability and intracellular signaling pathways, their application in mitochondrial study has been limited, largely due to the challenge of effectively and specifically expressing heterologous light-gated rhodopsin channels in the mitochondria. Here, we describe the methods for expressing functional channelrhodopsin 2 (ChR2) proteins in the mitochondrial inner membrane with an unusually long mitochondrial leading sequence and characterizing optogenetic-mediated mitochondrial membrane potential (ΔΨm) depolarization. We then illustrate how this next-generation optogenetic approach can be used to study the effect of ΔΨm on mitochondrial functions such as mitophagy, programed cell death, and preconditioning-mediated cytoprotection. We anticipate that this innovative technology will enable new insights into the mechanisms by which changes in ΔΨm differentially impacts mitochondrial and cellular functions.

Process improvement of adeno-associated virus (AAV) production.

Adeno-associated viruses (AAVs) have been well characterized and used to deliver therapeutic genes for diseases treatment in clinics and basic research. This study used the triple transient transfection of AAV-DJ/8 as a model expression system to develop and optimize the laboratory production of AAV for research and pre-clinical applications. Specifically, various production parameters, including host cell, transfection reagent, cell density, ratio of plasmid DNA and cells, gene size, and production mode, were tested to determine the optimal process. Our results showed that the adherent production using HEK 293AAV with calcium transfection generated the highest volumetric productivity of 7.86x109 gc/mL. The optimal suspensive production using HEK 293F had best AAV productivity of 5.78x109 gc/mL in serum-free medium under transfection conditions of transfection density of 0.4x106 cells/mL, plasmid DNA:cells ratio of 1.6 µg:106 cells and synthesized cationic liposomes as transfection reagent. The similar AAV productivity was confirmed at scales of 30 mL - 450 mL in shaker and/or spinner flasks. The in vitro transfection and in vivo infection efficiency of the harvested AAV-DJ/8 carrying luciferase reporter gene was confirmed using cell line and xenograft mouse model, respectively. The minimal or low purification recovery rate of AAV-DJ/8 in ion-exchange chromatography column and affinity column was observed in this study. In summary, we developed and optimized a scalable suspensive production of AAV to support the large-scale preclinical animal studies in research laboratories.

Anti-CD47 Monoclonal Antibody-Drug Conjugate: A Targeted Therapy to Treat Triple-Negative Breast Cancers.

Triple-negative breast cancers (TNBCs) are frequently recurrent due to the development of drug resistance post chemotherapy. Both the existing literature and our study found that surface receptor CD47 (cluster of differentiation 47) was upregulated in chemotherapy-treated TNBC cells. The goal of this study was to develop a monoclonal antibody (mAb)-based targeting strategy to treat TNBC after standard treatment. Specifically, a new mAb that targets the extracellular domain of receptor CD47 was developed using hybridoma technology and produced in fed-batch culture. Flow cytometry, confocal microscopy, and in vivo imaging system (IVIS) showed that the anti-CD47 mAb effectively targeted human and mouse TNBC cells and xenograft models with high specificity. The antibody-drug conjugate (ADC) carrying mertansine was constructed and demonstrated higher potency with reduced IC50 in TNBC cells than did the free drug and significantly inhibited tumor growth post gemcitabine treatment in MDA-MB-231 xenograft NSG model. Finally, whole blood analysis indicated that the anti-CD47 mAb had no general immune toxicity, flow cytometry analysis of lymph nodes revealed an increase of CD69+ NK, CD11c+ DC, and CD4+ T cells, and IHC staining showed tumoral infiltration of macrophage in the 4T1 xenograft BALB/cJ model. This study demonstrated that targeting CD47 with ADC has great potential to treat TNBCs as a targeted therapy.

Targeted Liposomal Chemotherapies to Treat Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBCs) are highly aggressive and recurrent. Standard cytotoxic chemotherapies are currently the main treatment options, but their clinical efficacies are limited and patients usually suffer from severe side effects. The goal of this study was to develop and evaluate targeted liposomes-delivered combined chemotherapies to treat TNBCs. Specifically, the IC50 values of the microtubule polymerization inhibitor mertansine (DM1), mitotic spindle assembly defecting taxane (paclitaxel, PTX), DNA synthesis inhibitor gemcitabine (GC), and DNA damage inducer doxorubicin (AC) were tested in both TNBC MDA-MB-231 and MDA-MB-468 cells. Then we constructed the anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) tagged liposomes and confirmed its TNBC cell surface binding using flow cytometry, internalization with confocal laser scanning microscopy, and TNBC xenograft targeting in NSG female mice using In Vivo Imaging System. The safe dosage of anti-EGFR liposomal chemotherapies, i.e., <20% body weight change, was identified. Finally, the in vivo anti-tumor efficacy studies in TNBC cell line-derived xenograft and patient-derived xenograft models revealed that the targeted delivery of chemotherapies (mertansine and gemcitabine) can effectively inhibit tumor growth. This study demonstrated that the targeted liposomes enable the new formulations of combined therapies that improve anti-TNBC efficacy.

Bioreactor Suspension Culture: Differentiation and Production of Cardiomyocyte Spheroids From Human Induced Pluripotent Stem Cells.

Human induced-pluripotent stem cells (hiPSCs) can be efficiently differentiated into cardiomyocytes (hiPSC-CMs) via the GiWi method, which uses small-molecule inhibitors of glycogen synthase kinase (GSK) and tankyrase to first activate and then suppress Wnt signaling. However, this method is typically conducted in 6-well culture plates with two-dimensional (2D) cell sheets, and consequently, cannot be easily scaled to produce the large numbers of hiPSC-CMs needed for clinical applications. Cell suspensions are more suitable than 2D systems for commercial biomanufacturing, and suspended hiPSCs form free-floating aggregates (i.e., spheroids) that can also be differentiated into hiPSC-CMs. Here, we introduce a protocol for differentiating suspensions of hiPSC spheroids into cardiomyocytes that is based on the GiWi method. After optimization based on cardiac troponin T staining, the purity of hiPSC-CMs differentiated via our novel protocol exceeded 98% with yields of about 1.5 million hiPSC-CMs/mL and less between-batch purity variability than hiPSC-CMs produced in 2D cultures; furthermore, the culture volume could be increased ∼10-fold to 30 mL with no need for re-optimization, which suggests that this method can serve as a framework for large-scale hiPSC-CM production.

Antibody-Drug Conjugate to Treat Meningiomas.

Meningiomas are primary tumors of the central nervous system with high recurrence. It has been reported that somatostatin receptor 2 (SSTR2) is highly expressed in most meningiomas, but there is no effective targeted therapy approved to control meningiomas. This study aimed to develop and evaluate an anti-SSTR2 antibody-drug conjugate (ADC) to target and treat meningiomas. The meningioma targeting, circulation stability, toxicity, and anti-tumor efficacy of SSTR2 ADC were evaluated using cell lines and/or an intracranial xenograft mouse model. The flow cytometry analysis showed that the anti-SSTR2 mAb had a high binding rate of >98% to meningioma CH157-MN cells but a low binding rate of <5% to the normal arachnoidal AC07 cells. The In Vivo Imaging System (IVIS) imaging demonstrated that the Cy5.5-labeled ADC targeted and accumulated in meningioma xenograft but not in normal organs. The pharmacokinetics study and histological analysis confirmed the stability and minimal toxicity. In vitro anti-cancer cytotoxicity indicated a high potency of ADC with an IC50 value of <10 nM. In vivo anti-tumor efficacy showed that the anti-SSTR2 ADC with doses of 8 and 16 mg/kg body weight effectively inhibited tumor growth. This study demonstrated that the anti-SSTR2 ADC can target meningioma and reduce the tumor growth.

Anti-EGFR antibody-drug conjugate for triple-negative breast cancer therapy.

Triple-negative breast cancers (TNBCs) are highly aggressive, metastatic and recurrent. Cytotoxic chemotherapies with limited clinical benefits and severe side effects are the standard therapeutic strategies, but, to date, there is no efficacious targeted therapy. Literature and our data showed that epidermal growth factor receptor (EGFR) is overexpressed on TNBC cell surface and is a promising oncological target. The objective of this study was to develop an antibody-drug conjugate (ADC) to target EGFR+ TNBC and deliver high-potency drug. First, we constructed an ADC by conjugating anti-EGFR monoclonal antibody with mertansine which inhibits microtubule assembly via linker Sulfo-SMCC. Second, we confirmed the TNBC-targeting specificity of anti-EGFR ADC by evaluating its surface binding and internalization in MDA-MB-468 cells and targeting to TNBC xenograft in subcutaneous mouse mode. The live-cell and live-animal imaging with confocal laser scanning microscopy and In Vivo Imaging System (IVIS) confirmed the TNBC-targeting. Finally, both in vitro toxicity assay and in vivo anti-cancer efficacy study in TNBC xenograft models showed that the constructed ADC significantly inhibited TNBC growth, and the pharmacokinetics study indicated its high circulation stability. This study indicated that the anti-EGFR ADC has a great potential to against TNBC.

Investigation into the difference in mitochondrial-cytosolic calcium coupling between adult cardiomyocyte and hiPSC-CM using a novel multifunctional genetic probe.

Ca2+ cycling plays a critical role in regulating cardiomyocyte (CM) function under both physiological and pathological conditions. Mitochondria have been implicated in Ca2+ handling in adult cardiomyocytes (ACMs). However, little is known about their role in the regulation of Ca2+ dynamics in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). In the present study, we developed a multifunctional genetically encoded Ca2+ probe capable of simultaneously measuring cytosolic and mitochondrial Ca2+ in real time. Using this novel probe, we determined and compared mitochondrial Ca2+ activity and the coupling with cytosolic Ca2+ dynamics in hiPSC-CMs and ACMs. Our data showed that while ACMs displayed a highly coordinated beat-by-beat response in mitochondrial Ca2+ in sync with cytosolic Ca2+, hiPSC-CMs showed high cell-wide variability in mitochondrial Ca2+ activity that is poorly coordinated with cytosolic Ca2+. We then revealed that mitochondrial-sarcoplasmic reticulum (SR) tethering, as well as the inter-mitochondrial network connection, is underdeveloped in hiPSC-CM compared to ACM, which may underlie the observed spatiotemporal decoupling between cytosolic and mitochondrial Ca2+ dynamics. Finally, we showed that knockdown of mitofusin-2 (Mfn2), a protein tethering mitochondria and SR, led to reduced cytosolic-mitochondrial Ca2+ coupling in ACMs, albeit to a lesser degree compared to hiPSC-CMs, suggesting that Mfn2 is a potential engineering target for improving mitochondrial-cytosolic Ca2+ coupling in hiPSC-CMs. Physiological relevance: The present study will advance our understanding of the role of mitochondria in Ca2+ handling and cycling in CMs, and guide the development of hiPSC-CMs for healing injured hearts.

Anti-SSTR2 antibody-drug conjugate for neuroendocrine tumor therapy.

Neuroendocrine (NE) tumors include a diverse spectrum of hormone-secreting neoplasms that arise from the endocrine and nervous systems. Current chemo- and radio-therapies have marginal curative benefits. The goal of this study was to develop an innovative antibody-drug conjugate (ADC) to effectively treat NE tumors (NETs). First, we confirmed that somatostatin receptor 2 (SSTR2) is an ideal cancer cell surface target by analyzing 38 patient-derived NET tissues, 33 normal organs, and three NET cell lines. Then, we developed a new monoclonal antibody (mAb, IgG1, and kappa) to target two extracellular domains of SSTR2, which showed strong and specific surface binding to NETs. The ADC was constructed by conjugating the anti-SSTR2 mAb and antimitotic monomethyl auristatin E. In vitro evaluations indicated that the ADC can effectively bind, internalize, release payload, and kill NET cells. Finally, the ADC was evaluated in vivo using a NET xenograft mouse model to assess cancer-specific targeting, tolerated dosage, pharmacokinetics, and antitumor efficacy. The anti-SSTR2 ADC exclusively targeted and killed NET cells with minimal toxicity and high stability in vivo. This study demonstrates that the anti-SSTR2 ADC has a high-therapeutic potential for NET therapy.

Targeted Exosomes for Drug Delivery: Biomanufacturing, Surface Tagging, and Validation.

Exosomes hold great potential to deliver therapeutic reagents for cancer treatment due to its inherent low antigenicity. However, several technical barriers, such as low productivity and ineffective cancer targeting, need to be overcome before wide clinical applications. The present study aims at creating a new biomanufacturing platform of cancer-targeted exosomes for drug delivery. Specifically, a scalable, robust, high-yield, cell line based exosome production process is created in a stirred-tank bioreactor, and an efficient surface tagging technique is developed to generate monoclonal antibody (mAb)-exosomes. The in vitro characterization using transmission electron microscopy, NanoSight, and western blotting confirm the high quality of exosomes. Flow cytometry and confocal laser scanning microscopy demonstrate that mAb-exosomes have strong surface binding to cancer cells. Furthermore, to validate the targeted drug delivery efficiency, romidepsin, a histone deacetylase inhibitor, is loaded into mAb-exosomes. The in vitro anti-cancer toxicity study shows high cytotoxicity of mAb-exosome-romidepsin to cancer cells. Finally, the in vivo study using tumor xenograft animal model validates the cancer targeting specificity, anti-cancer efficacy, and drug delivery capability of the targeted exosomes. In summary, new techniques enabling targeted exosomes for drug delivery are developed to support large-scale animal studies and to facilitate the translation from research to clinics.

Vector Analysis of Cytoskeletal Structural Tension and the Mechanisms that Underpin Spectrin-Related Forces in Pyroptosis.

Aims: Pyroptotic cells are characterized by plasma swelling, membrane blebbing, and disintegration of the cell membrane mediated by spectrin-based membrane skeleton and intercellular competitive tension activities. The spectrin-based membrane skeleton is involved in membrane organization through the regulation of intercellular tension. Using genetically encoded tension sensors to attain noninvasive force measurements in structural proteins, we investigated how cytoskeletal structural tension influences changes in plasma morphology during pyroptosis and the regulatory mechanism of cytoskeletal structural tension that underpins pyroptosis. Results: The results indicate that increasing spectrin tension is caused by osmotic swelling. Hightened tension of spectrin was closely associated with the shrink tension transmitted synergistically by microfilaments (MFs) and microtubules (MTs). However, the increment of spectrin tension in pyroptotic cells was controlled antagonistically by MF and MT forces. Different from MF tension, outward MT forces participated in the formation of membrane blebs. Spectrin tension caused by inward MF forces resisted pyroptosis swelling. Stabilization of MF and MT structure had little influence on intracellular tension and pyroptosis deformation. Pyroptosis-induced cytoskeletal structural tension was highly dependent on calcium signaling and reactive oxygen species generation. Blocking of membrane pores, nonselective ion flux, or elimination of caspase-1 cleavage resulted in the remission of structural forces associated with pyroptosis failure. Innovation and Conclusions: The data suggest that subcellular tension, in terms of magnitude and vector, is integral to pyroptosis through the mediation of swelling and blebbing and the elimination of structural tension, especially MT forces, may result in pyroptosis inhibition.

Comparative Proteomic Analysis of Three Chinese Hamster Ovary (CHO) Host Cells.

Chinese hamster ovary (CHO) cells have been widely used to express heterologous genes and produce therapeutic proteins in biopharmaceutical industry. Different CHO host cells have distinct cell growth rates and protein expression characteristics. In this study, the expression of about 1,307 host proteins in three sublines, i.e. CHO K1, CHO S and CHO/dihydrofolate reductase (dhfr)- , were investigated and compared using proteomic analysis. The proteins involved in cell growth, glycolysis, tricarboxylic acid cycle, transcription, translation and glycosylation were quantitated using Liquid chromatography tandem-mass spectrometry (LC-MS/MS). The key host cell proteins that regulate the kinetics of cell growth and the magnitude of protein expression levels were identified. Furthermore, several rational cell engineering strategies on how to combine the desired features of fast cell growth and efficient production of therapeutic proteins into one new super CHO host cell have been proposed.

Modulation effect of acidulated human serum albumin on Cu2+-mediated amyloid ß-protein aggregation and cytotoxicity under a mildly acidic condition.

Aggregation of amyloid ß-proteins (Aß) induced by Cu2+ is a crucial element in the pathogenesis of Alzheimer's disease (AD), and cerebral acidosis is a common complication of AD. Under mildly acidic conditions, Cu2+-Aß species have higher tendency to generate neurotoxic aggregates. Hence it is of significance to develop potent agents that inhibit Cu2+-mediated Aß aggregation under a mildly acidic condition. Herein we synthesized acidulated human serum albumin (A-HSA) to mitigate Cu2+-mediated Aß42 aggregation and cytotoxicity at pH6.6. Extensive experiments showed that A-HSA altered the pathway of Cu2+-mediated Aß42 aggregation and protected SH-SY5Y cells from cytotoxicity and oxidative damage induced by Cu2+-Aß42 species. Equimolar A-HSA increased cell viability from 52% to 91% as compared to Cu2+-Aß42-treated group. Stopped-flow fluorescence analysis revealed that A-HSA changed the Cu2+-Aß42 coordination mode from component I to II on the second timescale at pH6.6, which avoided the formation of aggregation-prone Cu2+-Aß42 aggregates. The findings revealed that the more negative charges on A-HSA surface could stabilize the protonated form of the adjacent histidine residues of Aß42. Hence, component I, which is necessary to form toxic aggregates, became unstable in the presence of A-HSA. On the other hand, hydrophobic binding and electrostatic repulsion could work simultaneously on the bound Aß42 on A-HSA surface. The two opposite forces stretched Aß42 conformations, which inhibited the formation of toxic Cu2+-Aß42 aggregates. Thus, A-HSA worked as a bifunctional inhibitor against Cu2+-mediated Aß42 aggregation and cytotoxicity under a mildly acidic condition.

Comparative proteomics analysis of high n-butanol producing metabolically engineered Clostridium tyrobutyricum.

The acidogenic Clostridium tyrobutyricum has recently been metabolically engineered to produce n-butanol. The objective of this study was to obtain a comprehensive understanding as to how butanol production was regulated in C. tyrobutyricum to guide the engineering of next-generation strains. We performed a comparative proteomics analysis, covering 78.1% of open reading frames and 95% of core enzymes, using wild type, ACKKO mutant (Δack) producing 37.30 g/L of butyrate and ACKKO-adhE2 mutant (Δack-adhE2) producing 16.68 g/L of butanol. In ACKKO-adhE2, the expression of most glycolytic enzymes was decreased, the thiolase (thl), acetyl-CoA acetyltransferase (ato), 3-hydroxybutyryl-CoA dehydrogenase (hbd) and crotonase (crt) that convert acetyl-CoA to butyryl-CoA were increased, and the heterologous bifunctional acetaldehyde/alcohol dehydrogenase (adhE2) catalyzing butanol formation was highly expressed. The apparent imbalance of energy and redox was observed due to the downregulation of acids production and the addition of butanol synthesis pathway, which also resulted in increased expression of chaperone proteins and glycerol-3-phosphate dehydrogenase (glpA) and the silence of sporulation transcription factor Spo0A (spo0A) as the cellular responses to butanol production. This study revealed the mechanism of carbon redistribution, and limiting factors and rational metabolic cell and process engineering strategies to achieve high butanol production in C. tyrobutyricum.

Characterization of novel mixed-mode protein adsorbents fabricated from benzoyl-modified polyethyleneimine-grafted Sepharose.

We have previously studied poly(ethylenimine) (PEI)-grafted Sepharose FF resins for ion-exchange chromatography of bovine serum albumin (BSA), and found the presence of a critical ionic capacity (cIC, 600mmol/L for BSA), above which both BSA adsorption capacity and uptake rates increased drastically. To extend the application of PEI-grafted resins, we have herein proposed to develop mixed-mode chromatographic (MMC) resins by modifying the grafted PEI chains with hydrophobic benzoyl groups. Three PEI-grafted resins with IC values from 329 to 701mmol/L (FF-PEI-L330, FF-PEI-L520 and FF-PEI-L700) were modified with benzoic acid. It was found that there was a maximum benzoyl density (BD) that could be reached for each resin, at which the average value of BD/residual IC was 1.2. The effect of BD (120-400mmol/L) on BSA adsorption (at pH 8.0) and elution (at pH 3.0) was first explored with FF-PEI-L700-derived resins. It was observed that both protein binding capacity and recovery increased with increasing BD, indicating that high BD was beneficial in protein adsorption. However, the elution of bound BSA with an acidic buffer (pH 3.0) was incomplete. It was hypothesized that the PEI chains, a pH-dependent cationic polyelectrolyte, formed a collapsed layer at the protein binding condition (high ionic strength, IS), while they exhibited extended spatial structures at elution (low pH and IS). These PEI chain structure transitions made the pores change from an opening state at the loading condition to a blocked state at the elution condition. The pore blocking was regarded as a chain-aroused-steric-hindrance (CaSH) effect. Thus, FF-PEI-L700 was not suitable for fabricating MMC resins due to its high chain density. Then, the effect of PEI density (the initial IC values of 330-700mmol/L) at the maximum BD values was investigated. Consequently, complete BSA recovery at pH 3.0 was obtained on the resin with an initial IC of 330mmol/L and a BD of 160mmol/L (B160-PEI330). The result indicates that the CaSH effect could be ignored at the low PEI chain density (IC=329mmol/L). Moreover, selective elution of γ-globulin could be achieved at pH 4.0 on the B160-PEI330 column, indicating the possibility of antibody purification from a mixture containing albumin by manipulating elution conditions. Finally, adsorption equilibria and uptake kinetics onto B160-PEI330 showed favorable binding properties for different proteins at a wide range of IS, indicating its usefulness as an MMC adsorbent.

Targeted biopharmaceuticals for cancer treatment.

Cancer is a complex invasive genetic disease that causes significant mortality rate worldwide. Protein-based biopharmaceuticals have significantly extended the lives of millions of cancer patients. This article reviews the biological function and application of targeted anticancer biopharmaceuticals. We first discuss the specific antigens and core pathways that are used in the development of targeted cancer therapy. The innovative monoclonal antibodies, non-antibody proteins, and small molecules targeting these antigens or pathways are then reviewed. Finally, the current challenges in anticancer biopharmaceuticals development and the potential solutions to address these challenges are discussed.