Probe-free optical chromatin deformation and measurement of differential mechanical properties in the nucleus.

The nucleus is highly organized to facilitate coordinated gene transcription. Measuring the rheological properties of the nucleus and its sub-compartments will be crucial to understand the principles underlying nuclear organization. Here, we show that strongly localized temperature gradients (approaching 1°C/µm) can lead to substantial intra-nuclear chromatin displacements (>1 µm), while nuclear area and lamina shape remain unaffected. Using particle image velocimetry (PIV), intra-nuclear displacement fields can be calculated and converted into spatio-temporally resolved maps of various strain components. Using this approach, we show that chromatin displacements are highly reversible, indicating that elastic contributions are dominant in maintaining nuclear organization on the time scale of seconds. In genetically inverted nuclei, centrally compacted heterochromatin displays high resistance to deformation, giving a rigid, solid-like appearance. Correlating spatially resolved strain maps with fluorescent reporters in conventional interphase nuclei reveals that various nuclear compartments possess distinct mechanical identities. Surprisingly, both densely and loosely packed chromatin showed high resistance to deformation, compared to medium dense chromatin. Equally, nucleoli display particularly high resistance and strong local anchoring to heterochromatin. Our results establish how localized temperature gradients can be used to drive nuclear compartments out of mechanical equilibrium to obtain spatial maps of their material responses.

Longitudinal in vivo imaging of perineuronal nets.

Significance: Perineuronal nets (PNNs) are extracellular matrix structures implicated in learning, memory, information processing, synaptic plasticity, and neuroprotection. However, our understanding of mechanisms governing the evidently important contribution of PNNs to central nervous system function is lacking. A primary cause for this gap of knowledge is the absence of direct experimental tools to study their role in vivo. Aim: We introduce a robust approach for quantitative longitudinal imaging of PNNs in brains of awake mice at subcellular resolution. Approach: We label PNNs in vivo with commercially available compounds and monitor their dynamics with two-photon imaging. Results: Using our approach, we show that it is possible to longitudinally follow the same PNNs in vivo while monitoring degradation and reconstitution of PNNs. We demonstrate the compatibility of our method to simultaneously monitor neuronal calcium dynamics in vivo and compare the activity of neurons with and without PNNs. Conclusion: Our approach is tailored for studying the intricate role of PNNs in vivo, while paving the road for elucidating their role in different neuropathological conditions.

Manufacturing a chimpanzee adenovirus-vectored SARS-CoV-2 vaccine to meet global needs.

Manufacturing has been the key factor limiting rollout of vaccination during the COVID-19 pandemic, requiring rapid development and large-scale implementation of novel manufacturing technologies. ChAdOx1 nCoV-19 (AZD1222, Vaxzevria) is an efficacious vaccine against SARS-CoV-2, based upon an adenovirus vector. We describe the development of a process for the production of this vaccine and others based upon the same platform, including novel features to facilitate very large-scale production. We discuss the process economics and the "distributed manufacturing" approach we have taken to provide the vaccine at globally-relevant scale and with international security of supply. Together, these approaches have enabled the largest viral vector manufacturing campaign to date, providing a substantial proportion of global COVID-19 vaccine supply at low cost.

Impact of Fc N-linked glycans on in vivo clearance of an immunoglobulin G1 antibody produced by NS0 cell line.

The heterogeneity of glycosylation on therapeutic monoclonal antibodies (mAbs) may affect the safety and efficacy of these agents. In particular, glycans of nonhuman origin, such as galactose-alpha-1,3-galactose (gal-α-gal) and N-glycolylneuraminic acid (NGNA), in the Fc region of therapeutic mAbs produced from murine cell lines carry a risk of immunogenicity. Immunogenic glycan structures can have immune-mediated clearance, resulting in faster clearance from in vivo circulation than non-immunogenic structures. To demonstrate the impact of these Fc nonhuman glycans on in vivo clearance, we purified and analyzed the glycan profile of a monoclonal antibody (mAb1) from human serum samples collected from clinical study participants. We purified mAb1 in a three-step chromatographic separation process (protein A, immobilized anti-mAb1 antibody affinity, and weak cation exchange chromatography) and extracted and labeled its N-linked oligosaccharide structures with 2-aminobenzamide acid for analysis on ultrahigh-performance hydrophilic interaction liquid chromatography. A comparison of the glycan profile of mAb1 recovered from human serum on the same day and 4 weeks after dosing revealed no significant differences, indicating similar clearance of mAb1 with nonhuman gal-α-gal or NGNA glycan in the Fc region compared with the human glycans. The relative proportions of the glycans remained similar, and all patients who had already received multiple doses of mAb1 over the course of a year were negative for antidrug antibodies, suggesting that none of the glycans induced an immune response. Therefore, we concluded that mAb1 gal-α-gal and NGNA glycoforms represent a low risk of conferring immunogenicity.

Prevention of Fab-arm exchange and antibody reduction via stabilization of the IgG4 hinge region.

IgG4s are dynamic molecules that undergo a process called Fab-arm exchange. Disulfide bonds between heavy chains are transiently reduced, resulting in half antibodies that reform intact antibodies with other IgG4 half antibodies. In vivo, therapeutic IgG4s can recombine with endogenous IgG4s, resulting in a heterogeneous mixture of bispecific antibodies. A related issue that can occur for any therapeutic protein during manufacturing is interchain disulfide bond reduction. For IgG4s, this primarily results in high levels of half-mAb that persist through purification processes. The S228P mutation has been used to prevent half-mAb formation. However, we demonstrated that IgG4s with the S228P mutation are subject to half-mAb formation and Fab-arm exchange in reducing environments. We identified two novel mutations that stabilize the heavy-heavy chain interaction via incorporation of additional disulfide bonds in the hinge region. Individually, these mutations increase stability toward reduction and lessen Fab-arm exchange. Combination of all three mutations, Y219C, G220C, and S228P, has an additive benefit resulting in an IgG4 with ˃7-fold increase in stability toward reduction while preventing Fab-arm exchange. Importantly, the mutations do not affect antigen binding or Fc effector function. These mutations hold great promise for solving mAb reduction during manufacturing and preventing Fab-arm exchange in vivo.

International Multicenter Survey of Perioperative Management of External Ventricular Drains: Results of the EVD Aware Study.

INTRODUCTION: The perioperative management of patients with external ventricular drains (EVDs) is not well defined, and adherence to published management guidelines unknown. This study investigates practice, patterns, and variability in the perioperative management of patients with EVDs. METHODS: A 31-question survey was sent to 1830 anesthesiologists from 27 institutions in North America, Europe, and Asia. A perioperative EVD Guideline Adherence Score was calculated for the preoperative, transport and intraoperative periods. Differences in management practices between neuroanesthesiologists and non-neuroanesthesiologists, and factors affecting EVD guideline adherence, were examined using bivariate significance tests and linear regression. RESULTS: Among a sample of 599 anesthesiologists (survey response rate, 32.7%), compared with non-neuroanesthesiologists, neuroanesthesiologists were more likely to include baseline neurological examination (P=0.023), hourly cerebrospinal fluid output (P=0.006) and color (P<0.001), intracranial pressure trends (P<0.001), and EVD clamp trial (P<0.001) data in their routine preanesthetic assessment of patients with EVDs. There was a low prevalence of routine intracranial pressure monitoring during patient transport of patients with EVDs (14.4%). Overall, 25.9% of respondents were aware of EVD guidelines, and 21% reported receiving formal training in EVD management. The EVD Guideline Adherence Score was highest among anesthesiologists who reported being very comfortable in managing patients with EVDs compared with those who reported being uncomfortable (9.93 vs. 6.93, P<0.001). CONCLUSIONS: The EVD Aware study identifies opportunities for improvement in the perioperative management of patients with EVDS, including global awareness, formal EVD training, and dissemination of educational tools.

Exosomes as a storehouse of tissue remodeling proteases and mediators of cancer progression.

Rapidly increasing scientific reports of exosomes and their biological effects have improved our understanding of their cellular sources and their cell-to-cell communication. These nano-sized vesicles act as potent carriers of regulatory bio-macromolecules and can induce regulatory functions by delivering them from its source to recipient cells. The details of their communication network are less understood. Recent studies have shown that apart from delivering its cargo to the cells, it can directly act on extracellular matrix (ECM) proteins and growth factors and can induce various remodeling events. More importantly, exosomes carry many surface-bound proteases, which can cleave different ECM proteins and carbohydrates and can shed cell surface receptors. These local extracellular events can modulate signaling cascades, which consequently influences the whole tissue and organ. This review aims to highlight the critical roles of exosomal proteases and their mechanistic insights within the cellular and extracellular environment.

Demystifying the extracellular matrix and its proteolytic remodeling in the brain: structural and functional insights.

The extracellular matrix (ECM) plays diverse roles in several physiological and pathological conditions. In the brain, the ECM is unique both in its composition and in functions. Furthermore, almost all the cells in the central nervous system contribute to different aspects of this intricate structure. Brain ECM, enriched with proteoglycans and other small proteins, aggregate into distinct structures around neurons and oligodendrocytes. These special structures have cardinal functions in the normal functioning of the brain, such as learning, memory, and synapse regulation. In this review, we have compiled the current knowledge about the structure and function of important ECM molecules in the brain and their proteolytic remodeling by matrix metalloproteinases and other enzymes, highlighting the special structures they form. In particular, the proteoglycans in brain ECM, which are essential for several vital functions, are emphasized in detail.

Multi-well Fibrillation Assay as a Tool for Analyzing Crosslinking and Stabilization of Collagen.

Collagen is the most widely used substratum in cell culture and biomaterials applications. In this chapter, we describe a simple procedure to isolate collagen, which can be employed to a wide range of tissue sources, and subsequently use it to study the collagen crosslinking and stabilization abilities of various compounds. The protocol is designed for a multi-well format assay and thus can be used for simultaneous assessment of multiple number of compounds and can be easily adapted to a high-throughput screening setup.

Brain Dynamics and Temporal Summation of Pain Predicts Neuropathic Pain Relief from Ketamine Infusion.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Ketamine is an N-methyl-D-aspartate receptor antagonist that reduces temporal summation of pain and modulates antinociception. Ketamine infusions can produce significant relief of neuropathic pain, but the treatment is resource intensive and can be associated with adverse effects. Thus, it is crucial to select patients who might benefit from this treatment. The authors tested the hypothesis that patients with enhanced temporal summation of pain and the capacity to modulate pain via the descending antinociceptive brain pathway are predisposed to obtain pain relief from ketamine. METHODS: Patients with refractory neuropathic pain (n = 30) and healthy controls underwent quantitative sensory testing and resting-state functional magnetic resonance imaging and then completed validated questionnaires. Patients then received outpatient intravenous ketamine (0.5 to 2 mg · kg · h; mean dose 1.1 mg · kg · h) for 6 h/day for 5 consecutive days. Pain was assessed 1 month later. Treatment response was defined as greater than or equal to 30% pain relief (i.e., reduction in pain scores). We determined the relationship between our primary outcome measure of pain relief with pretreatment temporal summation of pain and with brain imaging measures of dynamic functional connectivity between the default mode network and the descending antinociceptive brain pathway. RESULTS: Approximately 50% of patients achieved pain relief (mean ± SD; Responders, 61 ± 35%; Nonresponders, 7 ± 14%). Pretreatment temporal summation was associated with the effect of ketamine (ρ = -0.52, P = 0.003) and was significantly higher in Responders (median [25th, 75th] = 200 [100, 345]) compared with Nonresponders (44 [9, 92]; P = 0.001). Pretreatment dynamic connectivity was also associated with the clinical effect of ketamine (ρ = 0.51, P = 0.004) and was significantly higher in Responders (mean ± SD, 0.55 ± 0.05) compared with Nonresponders (0.51 ± 0.03; P = 0.006). Finally, the dynamic engagement of the descending antinociceptive system significantly mediated the relationship between pretreatment pain facilitation and pain relief (95% CI, 0.005 to 0.065). CONCLUSIONS: These findings suggest that brain and behavioral measures have the potential to prognosticate and develop ketamine-based personalized pain therapy.

Intracellular response to process optimization and impact on productivity and product aggregates for a high-titer CHO cell process.

A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters. Multiple process outputs were evaluated including cell growth, osmolality, lactate production, ammonium concentration, antibody production, and aggregate levels. Additionally, detailed assessment of oxygen uptake, nutrient and amino acid consumption, extracellular and intracellular redox environment, oxidative stress, activation of the unfolded protein response (UPR) pathway, protein disulfide isomerase (PDI) expression, and heavy and light chain mRNA expression provided an in-depth understanding of the cellular response to process changes. The results demonstrate that mRNA expression and UPR activation were unaffected by process changes, and that increased PDI expression and optimized nutrient supplementation are required for higher productivity processes. Furthermore, our findings demonstrate the role of extra- and intracellular redox environment on productivity and antibody aggregation. Processes using the optimized medium, with increased concentrations of redox modifying agents, had the highest overall specific productivity, reduced aggregate levels, and helped cells better withstand the high levels of oxidative stress associated with increased productivity. Specific productivities of different processes positively correlated to average intracellular values of total glutathione. Additionally, processes with the optimized media maintained an oxidizing intracellular environment, important for correct disulfide bond pairing, which likely contributed to reduced aggregate formation. These findings shed important understanding into how cells respond to process changes and can be useful to guide future development efforts to enhance productivity and improve product quality.

Matrix metalloproteinases: The sculptors of chronic cutaneous wounds.

Cutaneous wound healing is a complex mechanism with multiple processes orchestrating harmoniously for structural and functional restoration of the damaged tissue. Chronic non-healing wounds plagued with infection create a major healthcare burden and is one of the most frustrating clinical problems. Chronic wounds are manifested by prolonged inflammation, defective re-epithelialization and haphazard remodeling. Matrix metalloproteinases (MMPs) are zinc dependent enzymes that play cardinal functions in wound healing. Understanding the pathological events mediated by MMPs during wound healing may pave way in identifying novel drug targets for chronic wounds. Here, we discuss the functions and skewed regulation of different MMPs during infection and chronic tissue repair. This review also points out the potential of MMPs and their inhibitors as therapeutic agents in treating chronic wounds during distinct phases of the wound healing. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Production and characterization of active recombinant human factor II with consistent sialylation.

Coagulation factor II (prothrombin; FII) is the pre-proteolyzed precursor to thrombin in the coagulation cascade. It has 10 sites of gamma-carboxylation, which are required for its bioactivity, and is N-glycosylated at three of four putative sites. Production of recombinant human FII (rhFII) using a platform fed-batch process designed for monoclonal antibody production resulted in low levels of gamma-carboxylation and sialylation. There have not been any prior reports of successful process development and clinical manufacture of rhFII with optimal, consistent gamma-carboxylation and sialylation. In order to develop such a fed-batch process, various process parameters were evaluated to determine their impact on product quality. Process temperature and temperature shift timing were important for both sialic acid level and gamma-carboxyglutamate (Gla) level. In addition, vitamin K concentration and the type of surfactant used for preparation of vitamin K stock solution were also important for gamma carboxylation. A fed-batch study performed with various medium additives known to be involved in the N-glycosylation pathway, such as N-acetyl-d-mannosamine (ManNAc), galactose (Gal), dexamethasone, and manganese sulfate, increased the level of sialylation and enabled the elucidation of some potential bottlenecks in the sialylation pathway. The optimized process based on these studies yielded a reduction in the level of missing Gla by 0.4 moles per mole of rhFII in cell culture and a nearly threefold increase in sialic acid level. The process was successfully implemented at the 2000 L scale where a high Gla level and sialylation levels were achieved in all GMP lots. Biotechnol. Bioeng. 2017;114: 1991-2000. © 2017 Wiley Periodicals, Inc.

Dermatopontin augments angiogenesis and modulates the expression of transforming growth factor beta 1 and integrin alpha 3 beta 1 in endothelial cells.

Dermatopontin (DPT) is a matricellular protein with cardinal roles in cutaneous wound healing. The protein is also reported to be altered in various anomalies including cancer. The present study is aimed to unravel the role of DPT in angiogenesis which is imperative in many physiological and pathological processes. DPT's capabilities on promoting angiogenesis were assessed using various in vitro and ex vivo systems. The results indicated that DPT enhances cell motility and induces lamellipodia formation in endothelial cells. Additionally, we noticed that DPT stimulates tube formation in endothelial cells when plated on a matrigel substrate. However, it was observed that DPT had no effect on the proliferation of endothelial cells even at higher concentrations and prolonged treatment periods. Additional experiments on CAM and aortic arch assays apparently depicted that DPT promotes neovascularisation and tube sprouting, thus unraveling its prominent role in angiogenesis. Further, PCR analysis revealed that endothelial cells are devoid of DPT expression, but when exogenously supplied, modulates the expression of transforming growth factor ß1 and integrin α3ß1 which are reported to have crucial roles in endothelial cell behaviour during angiogenesis. In conclusion, DPT possess vital pro-angiogenic properties and thus retains promising therapeutic values in managing chronic wounds and cancer.

Perioperative Management of Adult Patients With External Ventricular and Lumbar Drains: Guidelines From the Society for Neuroscience in Anesthesiology and Critical Care.

External ventricular drains and lumbar drains are commonly used to divert cerebrospinal fluid and to measure cerebrospinal fluid pressure. Although commonly encountered in the perioperative setting and critical for the care of neurosurgical patients, there are no guidelines regarding their management in the perioperative period. To address this gap in the literature, The Society for Neuroscience in Anesthesiology & Critical Care tasked an expert group to generate evidence-based guidelines. The document generated targets clinicians involved in perioperative care of patients with indwelling external ventricular and lumbar drains.

Recent advancements in nanotechnological strategies in selection, design and delivery of biomolecules for skin regeneration.

Skin is a very complex organ and hence designing a bioengineered skin model replicating the essential physiological characteristics for replacing the diseased or damaged parts has been a challenging goal for many. Newer technologies for satisfying most of the criteria are being attempted with the copious efforts of biologists, engineers, physiologists, using multitude of features in combination. Amongst them nanotechnology based biomaterials have gained prominence owing to the enhanced pharmacokinetics, bio-distribution profile, extended half-life and reduced side effects. Designing a matrix that can be assimilated into the body during the regeneration and delivering the essential pharmacological agents in a temporal and spatially specific manner is a tremendous goal. This review essentially deals with the various approaches for designing a multidisciplinary translational smart matrix for addressing the various skin related ailments.

Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging.

Regulatory authorities require that cell lines used in commercial production of recombinant proteins must be derived from a single cell progenitor or clone. The limiting dilution method of cell cloning required multiple rounds of low-density cell plating and microscopic observation of a single cell in order to provide evidence of monoclonality. Other cloning methods rely on calculating statistical probability of monoclonality rather than visual microscopic observation of cells. We have combined the single cell deposition capability of the Becton Dickinson Influx™ cell sorter with the microscopic imaging capability of the SynenTec Cellavista to create a system for producing clonal production cell lines. The efficiency of single cell deposition by the Influx™ was determined to be 98% using fluorescently labeled cells. The centrifugal force required to settle the deposited cells to the bottom of the microplate well was established to be 1,126g providing a 98.1% probability that all cells will be in the focal plane of the Cellavista imaging system. The probability that a single cell was deposited by the cell sorter combined with the probability of every cell settling into the focal plane of the imager yield a combined >99% probability of documented monoclonality.

Fabrication of electrospun zein nanofibers for the sustained delivery of siRNA.

In this study, zein nanofibers based siRNA delivery system has been attempted for the first time. Here, the amphiphilic property of zein and the size advantage of nanofibers have been brought together in developing an ideal delivery system for siRNA. The morphological analysis of the GAPDH-siRNA loaded zein nanofibers revealed the proper encapsulation of the siRNA in the polymeric matrix. The loading efficiency of this delivery system was found to be 58.57±2.4% (w/w). The agarose gel analysis revealed that the zein nanofibers preserved the integrity of siRNA for a longer period even at the room temperature. The in vitro release studies not only depicted the sustaining potential of the zein nanofibers but also ensured the release of sufficient quantity of siRNA required to induce the gene silencing effect. The amphiphilic property of zein supported the cell attachment and thereby facilitated the transfection of siRNA into the cells. qRT-PCR analysis confirmed the potential of the developed system in inducing the desired gene silencing effect. Thus, electrospun zein nanofibers have been successfully employed for the delivery of siRNA which has a great therapeutic potential.