kDa Molecule Research Articles

Areca nut extract may be a potentially driving force for tumors to become autophagy addicted

We have previously described the mechanisms of action and composition of the autophagy-inducing ingredients found in areca nut (AN). These ingredients are 30–100 kDa molecules present in AN extract (ANE), referred to as ANE 30–100K. In other studies, we demonstrated that chronic stimulation with sub-lethal doses of ANE or ANE 30–100K resulted in increased tolerance to environmental challenges, including serum deprivation, hypoxic conditions, anti-cancer drugs, and accelerated tumor growth in nude mice, through upregulated autophagy activity. Such tumor cells were highly sensitive to the chemical inhibition of autophagy both in vitro and in vivo. Here, we further demonstrated similar inhibitory effects on the growth of stimulated CE81T/VGH cells in mice using atg5 shRNA. In contrast, the growth of non-stimulated control cells in mice was shown to be resistant to 3-methyladenine (3-MA). These findings suggest that autophagy blockade might be particularly effective for treating autophagy-dependent tumors in patients with AN-chewing habits.

Dynamics of scFv-targeted VAP2 correlating with IL-16, MIF and IL-1Ra in ANCA-associated vasculitis

Background and hypothesisUsing a mouse model of MPA with microvascular lesion with a clone (VasSF) of recombinant single chain fragments of the variable region of human IgG, we previously showed that vasculitis-associated apolipoprotein A2 (VAP2) may be a therapeutic target for vasculitis. The present study estimated the target molecules for VasSF and the association between VAP2 and cytokine levels in patient sera in terms of microvascular lesion severity. MethodsSera and clinical information were collected from patients with microscopic polyangiitis and granulomatosis with polyangiitis (MPA/GPA) and infectious disease. Neutrophil counts, levels of C-reactive protein (CRP), creatinine, total cholesterol associated with microvascular lesion, HDL cholesterol, low-density lipoprotein cholesterol, triglycerides, glomerular filtration rate (eGFR), and cytokines were estimated. Serum VAP2 signals were determined with Western blotting. ResultsVasSF bound to a 24 kDa molecule in the serum of active MPA/GPA patients. Anti-AP2 antibody also bound with the same 24 kDa molecule, named VAP2, because of size difference from normal APOA2. The VAP2 signal was significantly stronger in the active-disease group but significantly weakened in remission. The signal correlated positively with eGFR but not with the Birmingham Vasculitis Activity Score, CRP, MPO-ANCA, or PR3-ANCA levels. It correlated negatively with MPO activity, IL-16, MIF, and IL-1Ra. Moreover, VasSF bound to a 17 kDa molecule in the remission phase. ConclusionThe 24 kDa VAP2 molecule may be associated with neutrophil functions because of its inverse correlation with MPO activity, IL-16, MIF, and IL-1Ra, suggesting that VAP2-APOA1 formation in HDL triggers microvascular injury. VasSF may reverse the injury by removing APOA1-VAP2 heterodimers from peripheral blood vessels.

Distribution and transfer of organics in the coagulation–anaerobic hydrolysis–aerobic treatment process of cutting fluid wastewaters

ABSTRACT Two kinds of real cutting fluid wastewater samples (i.e., sample A and sample B) and two kinds of coagulants (i.e., polymerized aluminum chloride (PAC) and polymeric iron sulfate (PFS)) were studied during coagulation experiment, and the coagulation effluent of sample B with PAC was utilized for subsequent anaerobic hydrolysis–aerobic treatment, and the molecular weight (MW) distribution and ultraviolet (UV) absorption spectroscopy were used to explore the distribution and transfer of organics of each treatment unit. The results showed that sample A and sample B both mainly contained macromolecular organics with MW > 100 kDa and small molecule organics with MW < 3 kDa, and PAC had significant coagulation performance for organics with MW > 100 kDa in both samples, while neither PAC nor PFS achieved good coagulation performance for the small molecule organics with MW < 3 kDa in both samples. It could be inferred by UV spectra that the coagulation effluent of sample A and B might both contain organics with conjugated double bonds, monocyclic aromatic hydrocarbons, and the coagulation effluent of sample B might also contain polycyclic aromatic hydrocarbons and heterocyclic compounds with low MW. MW distribution analysis and the UV spectra results showed that biological treatment could further remove organics with conjugated double bonds and aromatic compounds with MW < 3 kDa.

Studying effective column lengths in liquid chromatography of large biomolecules

Based on their nature, large molecules tend to exhibit on-off elution such that only a small segment of a column bed participates in their separation. We were intrigued to investigate empirical data on this behavior and to apply a simple method to estimate the length of column bed that is needed to produce an effective separation. Models were derived by rearranging the linear solvent strength (LSS) model equations, and data sets from almost 100 different separation conditions were treated to illustrate effects for various types of solutes as separated by reversed phase (RP), ion-pair reversed phase (IP-RP), ion-exchange (IEX), hydrophobic interaction (HIC) and hydrophilic interaction (HILIC) chromatography.By empirically measuring S parameters (S is a solute dependent model parameter, it describes how sensitive is the solute retention to mobile phase composition), and calculating for an exit retention factor of 0.5, we have determined that there is little to no benefit to separating moderately sized solutes (5 – 10 kDa) with a column bed that is longer than 3 cm, particularly when a less than 20 min gradient is desired. Moreover, even shorter columns would be predicted to be adequate for 100 – 150 kDa molecules. Interpretations of this sort have become possible because there is some correlation between a solute's molecular weight and its S parameter. That is, empirical observations on retention behavior are not needed to select appropriate column lengths; molecular weight provides a sufficient approximation. With these insights, we suggest reconsidering the routine use of 5 – 15 cm long columns for >10 kDa biomolecule separations and instead propose that a new focus be placed on 1-2 cm long columns.

In silico Analysis of a 29 kDa Echinococcus granulosus Protoscolex Protein (P29) as a Vaccine Candidate against Cystic Echinococcosis.

Vaccination can be a key step in controlling hydatid cyst infection in humans and livestock in endemic areas of the disease. The aim of the Present study was to determine some of the basal biochemical properties followed by prediction and screening of B-cell and MHC-binding epitopes of EgP29 protein in silico. Some of the basic physico-chemical properties along with antigenicity, allergenicity, solubility, post-translational modification (PTM) sites, subcellular localization, signal peptide, transmembrane domain, secondary and tertiary structures followed by refinement and validations were computationally determined for this protein. Also, B-cell epitopes were predicted and screened using various web servers, while MHC-binding and CTL epitopes were predicted using IEDB and NetCTL servers, respectively. The protein is a 238-residue, 27 kDa molecule, with high thermotolerance (aliphatic: 71.81) and hydrophilicity (negative GRAVY). There were several glycosylation and phosphorylation sites in the sequence, without a transmembrane domain and signal peptide. Moreover, several B-cell and MHC-binding epitopes were found in the EgP29 protein, which could be further used in multi-epitope vaccines. In conclusion, results of the present study can be a promising sign for achieving effective approaches to the preparation of a multi-epitope vaccines against echinococcosis. So, it is necessary that the effectiveness of the protein and its epitopes be evaluated in vitro and in vivo.

Nanobubble-mediated cancer cell sonoporation using low-frequency ultrasound.

Ultrasound insonation of microbubbles can form transient pores in cell membranes that enable the delivery of non-permeable extracellular molecules to the cells. Reducing the size of microbubble contrast agents to the nanometer range could facilitate cancer sonoporation. This size reduction can enhance the extravasation of nanobubbles into tumors after an intravenous injection, thus providing a noninvasive sonoporation platform. However, drug delivery efficacy depends on the oscillations of the bubbles, the ultrasound parameters and the size of the target compared to the membrane pores. The formation of large pores is advantageous for the delivery of large molecules, however the small size of the nanobubbles limit the bioeffects when operating near the nanobubble resonance frequency at the MHz range. Here, we show that by coupling nanobubbles with 250 kHz low frequency ultrasound, high amplitude oscillations can be achieved, which facilitate low energy sonoporation of cancer cells. This is beneficial both for increasing the uptake of a specific molecule and to improve large molecule delivery. The method was optimized for the delivery of four fluorescent molecules ranging in size from 1.2 to 70 kDa to breast cancer cells, while comparing the results to targeted microbubbles. Depending on the fluorescent molecule size, the optimal ultrasound peak negative pressure was found to range between 300 and 500 kPa. Increasing the pressure to 800 kPa reduced the fraction of fluorescent cells for all molecules sizes. The optimal uptake for the smaller molecule size of 4 kDa resulted in a fraction of 19.9 ± 1.8% of fluorescent cells, whereas delivery of 20 kDa and 70 kDa molecules yielded 14 ± 0.8% and 4.1 ± 1.1%, respectively. These values were similar to targeted microbubble-mediated sonoporation, suggesting that nanobubbles can serve as noninvasive sonoporation agents with a similar potency, and at a reduced bubble size. The nanobubbles effectively reduced cell viability and may thus potentially reduce the tumor burden, which is crucial for the success of cancer treatment. This method provides a non-invasive and low-energy tumor sonoporation theranostic platform, which can be combined with other therapies to maximize the therapeutic benefits of cancer treatment or be harnessed in gene therapy applications.

Insight into the evolution characteristics on molecular weight of compost dissolved organic matters using high-performance size exclusion chromatography combined with a two-dimensional correlation analysis.

The information on molecular weight (MW) characteristics of DOM and relevant evolution behaviors during composting are limited. In this study, DOM extracted from co-composting of chicken manure and rice husks were comprehensively analyzed by using high-performance size exclusion chromatography (HPSEC) combined with a two-dimensional correlation spectroscopy (2D COS) to explore the evolution characteristics of MW of compost DOM. The HPSEC detected at UV of 254nm and at fluorescence (FL) Ex/Em wavelengths (315/410, 270/455nm) all showed a gradual increase in both weight-average and number-average MW for DOM, suggesting that the large MW fractions were continuously generated and polymerized during composting. The 2D COS applied on HPSEC-UV and -FL further identified the key active MW chromophoric (i.e., 0.5, 7.2. 9.5, 26.3, 30.7, and 83.9kDa) and fluorophoric (i.e., 0.55 and 3.5kDa) molecules that mainly participated in the transformation processes of compost DOM. Moreover, these active MW species were preferentially formed by the order of small to large molecules. A hetero-2D COS analysis disclosed the change sequence in the order of 0.5 and 7.2kDa chromophores → 3.5kDa fluorophores, and the 0.55 and 3.5kDa fluorophores → 26.3 and 83.9kDa chromophores.

The complexity of kidney disease and diagnosing it - cystatin C, selective glomerular hypofiltration syndromes and proteome regulation.

Estimation of kidney function is often part of daily clinical practice, mostly done by using the endogenous glomerular filtration rate (GFR)-markers creatinine or cystatin C. A recommendation to use both markers in parallel in 2010 has resulted in new knowledge concerning the pathophysiology of kidney disorders by the identification of a new set of kidney disorders, selective glomerular hypofiltration syndromes. These syndromes, connected to strong increases in mortality and morbidity, are characterized by a selective reduction in the glomerular filtration of 5-30kDa molecules, such as cystatin C, compared to the filtration of small molecules <1kDa dominating the glomerular filtrate, for example water, urea and creatinine. At least two types of such disorders, shrunken or elongated pore syndrome, are possible according to the pore model for glomerular filtration. Selective glomerular hypofiltration syndromes are prevalent in investigated populations, and patients with these syndromes often display normal measured GFR or creatinine-based GFR-estimates. The syndromes are characterized by proteomic changes promoting the development of atherosclerosis, indicating antibodies and specific receptor-blocking substances as possible new treatment modalities. Presently, the KDIGO guidelines for diagnosing kidney disorders do not recommend cystatin C as a general marker of kidney function and will therefore not allow the identification of a considerable number of patients with selective glomerular hypofiltration syndromes. Furthermore, as cystatin C is uninfluenced by muscle mass, diet or variations in tubular secretion and cystatin C-based GFR-estimation equations do not require controversial race or sex terms, it is obvious that cystatin C should be a part of future KDIGO guidelines.

Contribution of filtration and photocatalysis to DOM removal and fouling mechanism during in-situ UV-LED photocatalytic ceramic membrane process

The use of ceramic membranes and ultraviolet light-emitting diodes (UV-LEDs) has advanced the application of photocatalytic membrane for water treatment. We systematically evaluated the contribution of filtration and photocatalysis to dissolved organic matter (DOM) removal and fouling mechanism during in-situ UV-LED photocatalytic ceramic membrane filtration. The results showed that physical rejection primarily led to removal of 4–15 kDa molecules and photocatalysis further increased the removal of 1-4 kDa molecules, causing small sized microbial humic-like or protein-like materials in the permeate. In-situ UV-LED photocatalysis had an excellent effect on membrane fouling mitigation regardless of DOM sources. The dominant fouling mechanism changed from partial blockage to gel layer formation with increasing Ca2+ concentration but did not change with UV treatment. Correlation analysis revealed that the removal of 1-4 kDa molecules contributed to the mitigation of both reversible and irreversible fouling resistance, and the small molecules were the major cause of irreversible fouling resistance. Removal of 1–4 kDa terrestrial humic acid-like contributed to the pore blockage mechanism for synthetic water. Removal of 4-15 kDa protein-like materials was closely correlated to the pore blockage mechanism for real water. Trihalomethanes (THMs) and haloacetic acids (HAAs) formation potential (FP) were both significantly reduced after photocatalytic ceramic membrane process, but precursors of nitrogenous disinfection by-products (N-DBPs) with high toxicity were not removed by filtration or by photocatalysis, which deserves attention. Membrane rejection made higher contribution to better DBPFP control than photocatalysis. This study provides novel insights into the impact of UV-LED on DOM removal, DBPFP control and fouling mitigation, promoting the development of photocatalytic ceramic membrane filtration.

Efficacy optimization of low frequency microbubble-mediated sonoporation as a drug delivery platform to cancer cells

Ultrasound insonation of microbubbles can be used to form pores in cell membranes and facilitate the local trans-membrane transport of drugs and genes. An important factor in efficient delivery is the size of the delivered target compared to the generated membrane pores. Large molecule delivery remains a challenge, and can affect the resulting therapeutic outcomes. To facilitate large molecule delivery, large pores need to be formed. While ultrasound typically uses megahertz frequencies, it was recently shown that when microbubbles are excited at a frequency of 250 kHz (an order of magnitude below the resonance frequency of these agents), their oscillations are significantly enhanced as compared to the megahertz range. Here, to promote the delivery of large molecules, we suggest using this low frequency and inducing large pore formation through the high-amplitude oscillations of microbubbles. We assessed the impact of low frequency microbubble-mediated sonoporation on breast cancer cell uptake by optimizing the delivery of 4 fluorescent molecules ranging from 1.2 to 70 kDa in size. The optimal ultrasound peak negative pressure was found to be 500 kPa. Increasing the pressure did not enhance the fraction of fluorescent cells, and in fact reduced cell viability. For the smaller molecule sizes, 1.2 kDa and 4 kDa, the groups treated with an ultrasound pressure of 500 kPa and MB resulted in a fraction of 58% and 29% of fluorescent cells respectively, whereas delivery of 20 kDa and 70 kDa molecules yielded 10% and 5%, respectively. These findings suggest that low-frequency (e.g., 250 kHz) insonation of microbubbles results in high amplitude oscillation in vitro that increase the uptake of large molecules. Successful ultrasound-mediated molecule delivery requires the careful selection of insonation parameters to maximize the therapeutic effect by increasing cell uptake.

Endothelial Glycocalyx.

The glycocalyx is a polysaccharide structure that protrudes from the body of a cell. It is primarily conformed of glycoproteins and proteoglycans, which provide communication, electrostatic charge, ionic buffering, permeability, and mechanosensation-mechanotransduction capabilities to cells. In blood vessels, the endothelial glycocalyx that projects into the vascular lumen separates the vascular wall from the circulating blood. Such a physical location allows a number of its components, including sialic acid, glypican-1, heparan sulfate, and hyaluronan, to participate in the mechanosensation-mechanotransduction of blood flow-dependent shear stress, which results in the synthesis of nitric oxide and flow-mediated vasodilation. The endothelial glycocalyx also participates in the regulation of vascular permeability and the modulation of inflammatory responses, including the processes of leukocyte rolling and extravasation. Its structural architecture and negative charge work to prevent macromolecules greater than approximately 70 kDa and cationic molecules from binding and flowing out of the vasculature. This also prevents the extravasation of pathogens such as bacteria and virus, as well as that of tumor cells. Due to its constant exposure to shear and circulating enzymes such as neuraminidase, heparanase, hyaluronidase, and matrix metalloproteinases, the endothelial glycocalyx is in a continuous process of degradation and renovation. A balance favoring degradation is associated with a variety of pathologies including atherosclerosis, hypertension, vascular aging, metastatic cancer, and diabetic vasculopathies. Consequently, ongoing research efforts are focused on deciphering the mechanisms that promote glycocalyx degradation or limit its syntheses, as well as on therapeutic approaches to improve glycocalyx integrity with the goal of reducing vascular disease. © 2022 American Physiological Society. Compr Physiol 12: 1-31, 2022.

The Extracellular Chaperone Clusterin in Aβ and Non‐Aβ Cerebral Amyloidoses

Clusterin, a multifunctional glycoprotein also known as apolipoprotein J, has been implicated in several physiological processes and is an important contributor to many pathological conditions, including Alzheimer’s disease (AD) in which genetic variants and plasma levels of the apolipoprotein were identified as risk factors for the disorder. To investigate whether clusterin exerted a more general role in the pathophysiology of cerebral amyloidoses, we turned to two unrelated hereditary conditions, familial British and Danish dementias (FBD and FDD), which share striking neuropathological similarities with AD, including neurofibrillary degeneration in limbic areas as well as parenchymal and vascular amyloid deposition. Two different 4 kDa molecules – ABri in FBD and ADan in FDD – totally unrelated to the 4 kDa Alzheimer’s Aβ, are the main constituents of the amyloid deposits in these forms of cerebral amyloidoses. Mirroring findings in AD, a panel of immunohistochemical studies demonstrated clusterin co‐localization with cerebral parenchymal and vascular amyloid deposits in both disorders. Ligand affinity chromatography with downstream Western blot and amino acid sequence analyses unequivocally identified clusterin as the major ABri‐ and ADan‐binding plasma protein. Solid‐phase enzyme‐linked immunosorbent assays highlighted a specific saturable binding of clusterin to ABri and ADan. Non‐linear regression analysis of the data revealed Kd values within the same low nanomolar range as those demonstrated for the clusterin‐Aβ interaction. Consistent with its reported chaperone activity, clusterin showed a modulatory effect on ABri, ADan, and Aβ aggregation/fibrillization properties evaluated using thioflavin T, a dye that displays enhanced fluorescence upon binding fibrillar and protofibrillar amyloid conformations. Our findings, together with the known multifunctional activity of clusterin and its modulatory activity on the complex cellular pathways leading to oxidative stress, mitochondrial dysfunction, and the induction of cell death mechanisms – all known pathogenic features of protein folding disorders, including AD, FBD, and FDD – suggests the likelihood of a more complex role and a translational potential of clusterin in the amelioration/prevention of these pathogenic mechanisms.

Echinococcus granulosus cyclophilin: Immunoinformatics analysis to provide insights into the biochemical properties and immunogenic epitopes

Vaccination is a prominent strategy to confine cystic echinococcosis (CE) in areas of endemicity worldwide. The present study was performed to predict the primary biochemical features and immunogenic epitopes of Echinococcus granulosus cyclophilin protein as a potential vaccine candidate. Some of the basic physico-chemical properties along with antigenicity, allergenicity, solubility, post-translational modification (PTM) sites, subcellular localization, signal peptide, transmembrane domain, secondary and tertiary structures followed by refinement and validations were computationally determined for this protein. Also, B-cell epitopes were predicted and screened using various web servers, while MHC-binding and CTL epitopes were predicted using IEDB and NetCTL servers, respectively. The protein is a 162-residue, 17.35 kDa molecule, with relative thermotolerance (aliphatic: 60.12) and hydrophilicity (negative GRAVY). There were N-glycosylation and phosphorylation sites in the sequence, without a transmembrane domain. Moreover, several B-cell, MHC-binding and CTL epitopes were found in the EgGST protein, which could be further used in multi-epitope vaccines. In conclusion, the protein and its epitopes should be examined in wet lab experiments and in vivo.

Trappin-2/Elafin and Clusterin serum levels in pemphigus vulgaris and correlation with the severity score

Background Trappin-2/Elafin is a 9.9 KDa molecule released from its precursor Preproelafin that exists principally in immune cells, skin, the lungs, the vagina, and other organs. Clusterin is a heterodimeric glycoprotein that plays a major role in many biological processes such as interaction with lipids, apoptosis regulation, weakening of complement activation, toxin removal, response to damage, and stress as well as autoimmune damage. Both Trappin-2/Elafin and Clusterin serum levels have been studied in various immunologically mediated dermatological and nondermatological diseases. However, it still unknown whether their circulating levels are altered in pemphigus vulgaris (PV) and whether they play a role in this disease. Objective This study aimed to elucidate the potential link between both Trappin-2/Elafin and Clusterin levels and PV through a quantitative assessment of their serum levels by enzyme-linked immunosorbent assay and also to detect their possible correlations with PV severity using the pemphigus disease area index. Patients and methods Fifty patients with PV and 40 matched healthy controls were enrolled in this study. After a full assessment of history and complete dermatological examination, the severity score was calculated using pemphigus disease area index, and then serum samples were collected and subjected to quantitative measurements of serum Trappin-2/Elafin and Clusterin levels by enzyme-linked immunosorbent assay. Results Serum levels of both Trappin-2/Elafin and Clusterin were significantly higher in the patients than in the controls (P&lt;0.001); still, their levels were not correlated with the severity of the disease. Conclusion The finding indicates that both Trappin-2/Elafin and Clusterin serum levels become elevated in patients with PV; however, the increase is not specific for the disease. None of the markers are correlated with the severity score of PV. Increased Trappin-2/Elafin levels indicate the existence of chronic inflammation, autoimmunity and skin or other system damage. Increased Clusterin levels suggest autoimmune damage, stress or transforming growth factor stimulation.

Technical Note: Novel Use of CytoSorb™ Haemadsorption to Provide Wound Healing Support in Case of Severe Burn Trauma via Reduction of Hyperbilirubinaemia.

Hyperbilirubinaemia has been shown to compromise wound healing in severely burned patients. The therapy options for patients with impairment of wound healing and subsequent severe liver dysfunction are limited. A novel extracorporeal treatment, CytoSorb® (CytoSorbents Corp, USA), is a whole blood adsorber composed of highly biocompatible and porous polystyrene divinylbenzene copolymer beads covered in a polyvinylpyrrolidone coating. It is capable of extracting mainly hydrophobic middle-sized (up to 55 kDa) molecules from blood via size exclusion, including cytokines and bilirubin. We performed therapy with CytoSorb® on a severely burned (48% Total Body Surface Area-TBSA) patient with secondary sclerosing cholangitis (SCC) to promote the wound healing process by reducing bilirubin concentrations and to bridge the time to spontaneous liver regeneration or eventually to liver transplantation after two skin transplantations had failed to provide wound closure. In the first 6 days the cartridge was changed on a daily basis and later after every 2–4 days. The therapy with six adsorbers decreased a total bilirubin concentration from 14.02 to 4.29 mg/dl. By maintaining a stable bilirubin concentration under 5 mg/dl, debridement of abdomen and upper extremities with autologous skin grafting and, 4 weeks later, autologous skin grafting of the back from scrotum and lower extremities were performed successfully. After wound healing had been achieved, the CytoSorb therapy was discontinued after 57 days and 27 adsorber changes. CytoSorb therapy can be a promising support of wound and skin graft healing in patients with severe burns and liver dysfunction due to a significant reduction of total bilirubin concentration.

The Role of Adiponectin in the Skin.

Adiponectin (Ad), a 30 kDa molecule, is an anti-diabetic adipokine; although derived from adipose tissue, it performs numerous activities in various other tissues. It binds to its own receptors, namely adiponectin receptor 1(AdipoR1), adiponectin receptor 2 (AdipoR2), and T-cadherin (CDH13). Ad plays several roles, especially as a regulator. It modulates lipid and glucose metabolism and promotes insulin sensitivity. This demonstrates that Ad has a robust correlation with fat metabolism. Furthermore, although Ad is not in direct contact with other tissues, including the skin, it can be delivered to them by diffusion or secretion via the endocrine system. Recently it has been reported that Ad can impact skin cell biology, underscoring its potential as a therapeutic biomarker of skin diseases. In the present review, we have discussed the association between skin cell biology and Ad. To elaborate further, we described the involvement of Ad in the biology of various types of cells in the skin, such as keratinocytes, fibroblasts, melanocytes, and immune cells. Additionally, we postulated that Ad could be employed as a therapeutic target to maintain skin homeostasis.

Abstract 913: FLT3-targeting TriTACs are T cell engagers for treatment of acute myeloid leukemia

Abstract FLT3 (fms related receptor tyrosine kinase 3) is a cell surface protein expressed in hematopoietic stem and progenitor cell populations1. FLT3 RNA is overexpressed in greater than 95% of acute myeloid leukemia (AML) samples2, and FLT3 mutations are found in 25% to 35% of AML samples1. In 2020 in the United States, an estimated 19,940 new AML cases and 11,180 AML-related deaths are expected3, exemplifying that better therapies are needed to treat this disease. Given the frequency of FLT3 expression in AML and unmet medical need for patients, FLT3 represents an attractive target for T cell-redirecting immunotherapy in AML. FLT3-targeting Tri-specific T cell Activating Constructs (FLT3 TriTACs) are designed to simultaneously engage FLT3 on a target AML cell and CD3 on a T cell, resulting in T cell activation, proliferation, and lysis of the AML cell. FLT3 TriTACs are ~53 kDa molecules that consist of three binding domains: an N-terminal single domain antibody (sdAb) that binds to human FLT3, a middle sdAb that binds to human serum albumin (HSA) to extend the half-life, and a C-terminal single chain Fv (scFv) that binds to CD3ϵ of the T cell receptor (TCR). Biophysical analyses of these molecules indicate they are stable with high monomer content. FLT3 TriTACs bind to human and cynomolgus monkey FLT3, CD3ϵ and albumin with similar affinities. Flow cytometry analysis of T cells from various normal donors and a panel of FLT3-positive and FLT3-negative tumor cell lines confirmed binding of FLT3 TriTACs to their targets expressed on the cell surface. FLT3 TriTACs induce potent killing of FLT3-expressing AML cell lines in vitro. In co-cultures of T cells from normal human donors, target tumor cells, and HSA, these TriTACs mediated dose-dependent and FLT3-dependent cytotoxicity. Cell lines engineered to express cynomolgus FLT3 were also readily killed by T cells with FLT3 TriTACs. Preclinical evaluation of FLT3 TriTACs in vitro, in mouse tumor models, and cynomolgus monkey pharmacokinetic studies will be presented.

Backbone assignment of crystalline E. coli maltose binding protein.

The E.coli maltose binding protein (MBP) is a 42.5kDa molecule widely employed in many biotechnology applications. Because of its molecular size, it has become the main model system for the development of solution NMR methods adapted to large biomolecular targets. Here, we report virtually complete (~ 90%) backbone resonance assignments obtained on a microcrystalline sample of MBP with 1H-detected solid-state NMR at fast (> 100kHz) magic-angle spinning. We additionally present the detailed description of the methodology employed for the preparation of the sample and the acquisition and analysis of the NMR spectra. The chemical shifts, obtained with a single uniformly 15N,13C-labelled and fully-protonated sample and about 2 weeks on a 800MHz NMR spectrometer, have been deposited to the BMRB under the accession number 50089.

Glomerular filtration and shrunken pore syndrome in children and adults

A major function of the kidney is to, by glomerular filtration, maintain the overall steady-state of 5-30kDa proteins, many of which are signalling molecules. This function of the kidney has been overlooked, since predominantly low-molecular-mass substances <1kDa have been used to measure or estimate glomerular filtration rate (GFR). The use of cystatin C (13kDa) as a marker of GFR has allowed the discovery that the filtration of 5-30kDa molecules can be selectively impaired defining the shrunken pore syndrome. The discovery, pathophysiology, morbidity (mainly cardiovascular manifestations) and mortality of this syndrome are described.

A novel cell permeability assay for macromolecules

BackgroundMany cell permeabilisation methods to mediate internalisation of various molecules to mammalian or bacterial cells have been developed. However, no size-specific permeability assay suitable for both cell types exists.ResultsWe report the use of intrinsically biotinylated cell components as the target for reporter molecules for assessing permeabilisation. Due to its well-described biotin binding activity, we developed an assay using Streptavidin (SAv) as a molecular weight marker for assessing eukaryotic and prokaryotic cell internalisation, using flow cytometry as a readout. This concept was tested here as part of the development of host DNA depletion strategies for microbiome analysis of formalin-fixed (FF) samples. Host depletion (HD) strategies require differential cell permeabilisation, where mammalian cells but not bacterial cells are permeabilised, and are subsequently treated with a nuclease. Here, the internalisation of a SAv-conjugate was used as a reference for nucleases of similar dimensions. With this assay, it was possible to demonstrate that formalin fixation does not generate pores which allow the introduction of 60 KDa molecules in mammalian or bacterial membranes/envelopes. Among surfactants tested, Saponin derived from Quillaja bark showed the best selectivity for mammalian cell permeabilisation, which, when coupled with Benzonase nuclease, provided the best results for host DNA depletion, representing a new HD strategy for formalin fixed samples.ConclusionThe assay presented provides researchers with a sensitive and accessible tool for discerning membrane/cell envelop permeability for different size macromolecules.